2&#39; -modified oligonucleotides

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to modulation of the production of selected proteins. In accordance with preferred embodiments, oligonucleotides and oligonucleotide analogs are provided which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the 2′-deoxyfuranosyl moieties of the nucleoside unit is modified. Treatment of diseases caused by various viruses and other causative agents is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/468,037, filed on Jun. 6, 1995, which is acontinuation-in-part of U.S. patent application Ser. No. 07/835,932 (nowU.S. Pat. No. 5,670,633), filed Mar. 5, 1992 [which is the U.S. nationalphase application of International application PCT/US91/05720, filedAug. 12, 1991 (now abandoned)], which is a continuation-in-part of U.S.patent application Ser. No. 07/566,977, filed Aug. 13, 1990 (nowabandoned). This application is also a continuation-in-part of U.S.patent application Ser. No. 07/854,634 (now abandoned), filed Jul. 1,1992 [which is the U.S. national phase application of Internationalapplication PCT/US91/00243, filed Jan. 11, 1991 (now abandoned)], whichis a continuation-in-part of U.S. patent application Ser. No.07/463,358, filed Jan. 11, 1990 (now abandoned), and U.S. patentapplication Ser. No. 07/566,977, filed Aug. 13, 1990 (now abandoned).Each of the above-mentioned applications is commonly assigned with thisapplication, and the entire disclosures of each are herein incorporatedby reference.

FIELD OF THE INVENTION

[0002] This invention is directed to nuclease resistant oligonucleotideswhich are useful as therapeutics, diagnostics, and research reagents.Sugar-modified oligonucleotides which are resistant to nucleasedegradation and are capable of modulating the activity of DNA and RNAare provided.

BACKGROUND OF THE INVENTION

[0003] It has been recognized that oligonucleotides can be used tomodulate mRNA expression by a mechanism that involves the complementaryhybridization of relatively short oligonucleotides to mRNA such that thenormal, essential functions of these intracellular nucleic acids aredisrupted. Hybridization is the sequence-specific base pair hydrogenbonding of an oligonucleotide to a complementary RNA or DNA.

[0004] One deficiency of oligonucleotides for these purposes is theirsusceptibility to enzymatic degradation by a variety of ubiquitousnucleases which may be intracellularly and extracellularly located.Unmodified, “wild type”, oligonucleotides are not useful as therapeuticagents because they are rapidly degraded by nucleases. Therefore,modification of oligonucleotides for conferring nuclease resistance onthem has been a focus of research directed towards the development ofoligonucleotide therapeutics and diagnostics.

[0005] In addition to nuclease stability, the ability of anoligonucleotide to bind to a specific DNA or RNA with fidelity is afurther important factor.

[0006] The relative ability of an oligonucleotide to bind tocomplementary nucleic acids is compared by determining the meltingtemperature of a particular hybridization complex. The meltingtemperature (T_(m)), a characteristic physical property of doublehelices, is the temperature (in ° C.) at which 50% helical versus coil(unhybridized) forms are present. T_(m) is measured by using UVspectroscopy to determine the formation and breakdown (melting) ofhybridization. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m) the greater the strength of the binding of the nucleicacid strands.

[0007] Therefore, oligonucleotides modified to exhibit resistance tonucleases and to hybridize with appropriate strength and fidelity to itstargeted RNA (or DNA) are greatly desired for use as research reagents,diagnostic agents and as oligonucleotide therapeutics. Various2′-substitutions have been introduced in the sugar moiety ofoligonucleotides. The nuclease resistance of these compounds has beenincreased by the introduction of 2′-substituents such as halo, alkoxyand allyloxy groups.

[0008] Ikehara et al. [European Journal of Biochemistry 139, 447 (1984)]have reported the synthesis of a mixed octamer containing one2′-deoxy-2′-fluoroguanosine residue or one 2′-deoxy-2′-fluoroadenineresidue. Guschlbauer and Jankowski [Nucleic Acids Res. 8, 1421 (1980)]have shown that the contribution of the 3′-endo increases withincreasing electronegativity of the 2′-substituent. Thus,2′-deoxy-2′-fluorouridine contains 85% of the C3′-endo conformer.

[0009] Furthermore, evidence has been presented which indicates that2′-substituted-2′-deoxyadenosine polynucleotides resembledouble-stranded RNA rather than DNA. Ikehara et al. [Nucleic Acids Res.,5, 3315 (1978)] have shown that a 2′-fluoro substituent in poly A, polyI, or poly C duplexed to its complement is significantly more stablethan the ribonucleotide or deoxyribonucleotide poly duplex as determinedby standard melting assays. Ikehara et al. [Nucleic Acids Res., 4, 4249(1978)] have shown that a 2′-chloro or bromo substituent inpoly(2′-deoxyadenylic acid) provides nuclease resistance. Eckstein etal. [Biochemistry, 11, 4336 (1972)] have reported thatpoly(2′-chloro-2′-deoxyuridylic acid) andpoly(2′-chloro-2′-deoxycytidylic acid) are resistant to variousnucleases. Inoue et al. [Nucleic Acids Research, 15, 6131 (1987)] havedescribed the synthesis of mixed oligonucleotide sequences containing2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substitutedoligonucleotide hybridized to its RNA complement as strongly as theRNA-RNA duplex which is significantly stronger than the same sequenceRNA-DNA heteroduplex (T_(m)s, 49.0 and 50.1 versus 33.0 degrees fornonamers). Shibahara et al. [Nucleic Acids Research, 17, 239 (1987)]have reported the synthesis of mixed oligonucleotides containing 2′-OMesubstituents on every nucleotide. The mixed 2′-OMe-substitutedoligonucleotides were designed to inhibit HIV replication.

[0010] It is believed that the composite of the hydroxyl group's stericeffect, its hydrogen bonding capabilities, and its electronegativityversus the properties of the hydrogen atom is responsible for the grossstructural difference between RNA and DNA. Thermal melting studiesindicate that the order of duplex stability (hybridization) of2′-methoxy oligonucleotides is in the order of RNA-RNA>RNA-DNA>DNA-DNA.

[0011] U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixedoligonucleotides comprising an RNA portion, bearing 2′-O-alkylsubstituents, conjugated to a DNA portion via a phosphodiester linkage.However, being phosphodiesters, these oligonucleotides are susceptibleto nuclease cleavage.

[0012] European Patent application 339,842, filed Apr. 13, 1989,discloses 2′-O-substituted phosphorothioate oligonucleotides, including2′-O-methylribooligonucleotide phosphorothioate derivatives. Thisapplication also discloses 2′-O-methyl phosphodiester oligonucleotideswhich lack nuclease resistance.

[0013] European Patent application 260,032, filed Aug. 27, 1987,discloses oligonucleotides having 2′-O-methyl substituents on the sugarmoiety. This application also makes mention of other 2′-O-alkylsubstituents, such as ethyl, propyl and butyl groups.

[0014] International Publication Number WO 91/06556, published May 16,1991, discloses oligomers derivatized at the 2′ position withsubstituents, which are stable to nuclease activity. Specific2′-O-substituents which were incorporated into oligonucleotides includeethoxycarbonylmethyl (ester form), and its acid, amide and substitutedamide forms.

[0015] European Patent application 399,330, filed May 15, 1990,discloses nucleotides having 2′-O-alkyl substituents.

[0016] International Publication Number WO 91/15499, published Oct. 17,1991, discloses oligonucleotides bearing 2′-O-alkyl, -alkenyl and-alkynyl substituents.

[0017] It has been recognized that nuclease resistance ofoligonucleotides and fidelity of hybridization are of great importancein the development of oligonucleotide therapeutics. Oligonucleotidespossessing nuclease resistance are also desired as research reagents anddiagnostic agents.

BRIEF DESCRIPTION OF THE INVENTION

[0018] In accordance with the present invention, compositions which areresistant to nuclease degradation and those that modulate the activityof DNA and RNA are provided. These compositions are comprised ofsugar-modified oligonucleotides, which are specifically hybridizablewith preselected nucleotide sequences of single-stranded ordouble-stranded target DNA or RNA. The sugar-modified oligonucleotidesrecognize and form double strands with single-stranded DNA and RNA.

[0019] The nuclease resistant oligonucleotides of the present inventionconsist of a single strand of nucleic acid bases linked together throughlinking groups. The oligonucleotides of this invention may range inlength from about 5 to about 50 nucleic acid bases. However, inaccordance with a preferred embodiment of this invention, a sequence ofabout 12 to 25 bases in length is optimal.

[0020] The individual nucleotides of the oligonucleotides of the presentinvention are connected via phosphorus linkages. Preferred phosphorouslinkages include phosphodiester, phosphorothioate and phosphorodithioatelinkages, with phosphodiester and phosphorothioate linkages beingparticularly preferred.

[0021] Preferred nucleobases of the invention include adenine, guanine,cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine,8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substitutedguanines, other aza and deaza uracils, other aza and deaza thymidines,other aza and deaza cytosines, other aza and deaza adenines, other azaand deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

[0022] In accordance with this invention at least one of the2′-deoxyribofuranosyl moiety of at least one of the nucleosides of anoligonucleotide is modified. A halo, alkoxy, aminoalkoxy, alkyl, azido,or amino group may be added. For example, F, CN, CF₃, OCF₃, OCN,O-alkyl, S-alkyl, SMe, SO₂Me, ONO₂, NO₂, NH₃, NH₂, NH-alkyl, OCH₂CH═CH₂(allyloxy), OCH₃═CH₂, OCCH, where alkyl is a straight or branched chainof C₁ to C₂₀, with unsaturation within the carbon chain.

[0023] The present invention also includes oligonucleotides formed froma plurality of linked-β-nucleosides including2′-deoxy-erythro-pentofuranosyl-β-nucleosides. These nucleosides areconnected by charged phosphorus linkages in a sequence that isspecifically hybridizable with a complementary target nucleic acid. Thesequence of linked nucleosides is divided into at least twosubsequences. The first subsequence includes β-nucleosides, having2′-substituents, linked by charged 3′-5′ phosphorous linkages. Thesecond subsequence consists of2′-deoxy-erythro-pentofuranosyl-β-nucleosides linked by charged 3′-5′phosphorous linkages bearing a negative charge at physiological pH. Infurther preferred embodiments there exists a third subsequence whosenucleosides are selected from those selectable for the firstsubsequence. In preferred embodiments the second subsequence ispositioned between the first and third subsequences. Sucholigonucleotides of the present invention are also referred to as“chimeric” or “gapped” oligonucleotides, or “chimeras.”

[0024] The resulting novel oligonucleotides of the invention areresistant to nuclease degradation and exhibit hybridization propertiesof higher quality relative to wild-type DNA-DNA and RNA-DNA duplexes andphosphorus-modified oligonucleotide duplexes containingmethylphosphonates, phophoramidates and phosphate triesters.

[0025] The invention is also directed to methods for modulating theproduction of a protein by an organism comprising contacting theorganism with a composition formulated in accordance with the foregoingconsiderations. It is preferred that the RNA or DNA portion which is tobe modulated be preselected to comprise that portion of DNA or RNA whichcodes for the protein whose formation is to be modulated. Therefore, theoligonucleotide to be employed is designed to be specificallyhybridizable to the preselected portion of target DNA or RNA.

[0026] This invention is also directed to methods of treating anorganism having a disease characterized by the undesired production of aprotein. This method comprises contacting the organism with acomposition in accordance with the foregoing considerations. Thecomposition is preferably one which is designed to specifically bindwith mRNA which codes for the protein whose production is to beinhibited.

[0027] The invention further is directed to diagnostic methods fordetecting the presence or absence of abnormal RNA molecules, or abnormalor inappropriate expression of normal RNA molecules in organisms orcells.

[0028] The invention is also directed to methods for the selectivebinding of RNA for use as research reagents and diagnostic agents. Suchselective and strong binding is accomplished by interacting such RNA orDNA with oligonucleotides of the invention which are resistant todegradative nucleases and which display greater fidelity ofhybridization than any other known oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a graph showing dose response activity ofoligonucleotides of the invention and a reference compound.

[0030]FIG. 2 is a bar chart showing dose response activity ofoligonucleotides of the invention and reference compounds.

[0031]FIG. 3 is a bar graph showing the effects of several 2′-O-methylchimeric oligonucleotides on PKC-α mRNA levels. Hatched bars representthe 8.5 kb transcript, and plain bars represent the 4.0 kb transcript.

[0032]FIG. 4 is a bar graph showing the effects of several 2′-O-methyland 2′-O-propyl chimeric oligonucleotides on PKC-α mRNA levels. Hatchedbars represent the 8.5 kb transcript, and plain bars represent the 4.0kb transcript.

[0033]FIG. 5 is a bar graph showing the effects of additional2′-O-methyl and 2′-O-propyl chimeric oligonucleotides on PKC-α mRNAlevels. Hatched bars represent the 8.5 kb transcript, and plain barsrepresent the 4.0 kb transcript.

[0034]FIG. 6 is a graph showing mouse plasma concentrations of a controlcompound and two of the compounds of the invention. The plasmaconcentration is plotted verses time.

[0035]FIG. 7 is a three dimensional graph showing distribution of acontrol compound among various tissue in the mouse. Specific tissues areshown on one axis, time on a second axis and percent of dose on thethird axis. The compound was delivered by intravenous injected.

[0036]FIG. 8 is a three dimensional graph showing distribution of acompound of the invention among various tissue in the mouse. Specifictissues are shown on one axis, time on a second axis and percent of doseon the third axis. The compound was delivered by intravenous injected.

[0037]FIG. 9 is a three dimensional graph showing distribution of afurther compound of the invention among various tissue in the mouse.Specific tissues are shown on one axis, time on a second axis andpercent of dose on the third axis. The compound was delivered byintravenous injected.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0038] The compositions useful for modulating the activity of an RNA orDNA molecule in accordance with this invention generally comprise asugar-modified oligonucleotide which is specifically hybridizable with apreselected nucleotide sequence of a single-stranded or double-strandedtarget DNA or RNA molecule, and which is nuclease resistant.

[0039] It is generally desirable to select a sequence of DNA or RNAwhich is involved in the production of a protein whose synthesis isultimately to be modulated or inhibited in its entirety. Theoligonucleotides of the invention are conveniently synthesized usingsolid phase synthesis of known methodology, and is designed to becomplementary to or specifically hybridizable with the preselectednucleotide sequence of the target RNA or DNA. Nucleic acid synthesizersare commercially available and their use is understood by persons ofordinary skill in the art as being effective in generating any desiredoligonucleotide of reasonable length.

[0040] The oligonucleotides of the invention also include those thatcomprise nucleosides connected by charged linkages, and whose sequencesare divided into at least two subsequences. The first subsequenceincludes 2′-substituted-nucleosides linked by a first type of linkage.The second subsequence includes nucleosides linked by a second type oflinkage. In a preferred embodiment there exists a third subsequencewhose nucleosides are selected from those selectable for the firstsubsequence, and the second subsequence is positioned between the firstand the third subsequences. Such oligonucleotides of the invention areknown as “chimeras,” or “chimeric” or “gapped” oligonucleotides.

[0041] In the context of this invention, the term “oligonucleotide”refers to a plurality of nucleotides joined together in a specificsequence from naturally and non-naturally occurring nucleobases.Preferred nucleobases of the invention are joined through a sugar moietyvia phosphorus linkages, and include adenine, guanine, adenine,cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine,8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substitutedguanines, other aza and deaza uracils, other aza and deaza thymidines,other aza and deaza cytosines, other aza and deaza adenines, other azaand deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.The sugar moiety may be deoxyribose or ribose. The oligonucleotides ofthe invention may also comprise modified nucleobases or nucleobaseshaving other modifications consistent with the spirit of this invention,and in particular modifications that increase their nuclease resistancein order to facilitate their use as therapeutic, diagnostic or researchreagents.

[0042] The oligonucleotides of the present invention are about 5 toabout 50 bases in length. It is more preferred that the oligonucleotidesof the invention have from 8 to about 40 bases, and even more preferredthat from about 12 to about 25 bases be employed.

[0043] It is desired that the oligonucleotides of the invention beadapted to be specifically hybridizable with the nucleotide sequence ofthe target RNA or. DNA selected for modulation. Oligonucleotidesparticularly suited for the practice of one or more embodiments of thepresent invention comprise 2′-sugar modified oligonucleotides whereinone or more of the 2′-deoxy ribofuranosyl moieties of the nucleoside ismodified with a halo, alkoxy, aminoalkoxy, alkyl, azido, or amino group.For example, the substitutions which may occur include F, CN, CF₃, OCF₃,OCN, O-alkyl, S-alkyl, SMe, SO₂Me, ONO₂, NO₂, NH₃, NH₂, NH-alkyl,OCH₃═CH₂ and OCCH. In each of these, alkyl is a straight or branchedchain of C₁ to C₂₀, having unsaturation within the carbon chain. Apreferred alkyl group is C₁-C₉ alkyl. A further preferred alkyl group isC₅-C₂₀ alkyl.

[0044] A first preferred group of substituents include2′-deoxy-2′-fluoro substituents. A further preferred group ofsubstituents include C₁-C₂₀ alkoxyl substituents. An additionalpreferred group of substituents include cyano, fluoromethyl,thioalkoxyl, fluoroalkoxyl, alkylsulfinyl, alkylsulfonyl, allyloxy andalkeneoxy substituents.

[0045] In further embodiments of the present invention, the individualnucleotides of the oligonucleotides of the invention are connected viaphosphorus linkages. Preferred phosphorus linkages includephosphodiester, phosphorothioate and phosphorodithioate linkages. In onepreferred embodiment of this invention, nuclease resistance is conferredon the oligonucleotides by utilizing phosphorothioate internucleosidelinkages.

[0046] In further embodiments of the invention, nucleosides can bejoined via linkages that substitute for the internucleoside phosphatelinkage. Macromolecules of this type have been identified asoligonucleosides. The term “oligonucleoside” thus refers to a pluralityof nucleoside units joined by non-phosphorus linkages. In sucholigonucleosides the linkages include an —O—CH₂—CH₂—O— linkage (i.e., anethylene glycol linkage) as well as other novel linkages disclosed inU.S. Pat. No. 5,223,618, issued Jun. 29, 1993, U.S. Pat. No. 5,378,825,issued Jan. 3, 1995 and U.S. patent application Ser. No. 08/395,168,filed Feb. 27, 1995. Other modifications can be made to the sugar, tothe base, or to the phosphate group of the nucleotide. Representativemodifications are disclosed in International Publication Numbers WO91/10671, published Jul. 25, 1991, WO 92/02258, published Feb. 20, 1992,WO 92/03568, published Mar. 5, 1992, and U.S. Pat. No. 5,138,045, issuedAug. 11, 1992, all assigned to the assignee of this application. Thedisclosures of each of the above referenced publications are hereinincorporated by reference.

[0047] In the context of this invention, “hybridization” shall meanhydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. “Complementary,” as usedherein, also refers to sequence complementarity between two nucleotides.For example, if a nucleotide at a certain position of an oligonucleotideis capable of hydrogen bonding with a nucleotide at the same position ofa DNA or RNA molecule, then the oligonucleotide and the DNA or RNA areconsidered to be complementary to each other at that position. Theoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the oligonucleotide and the DNA or RNAtarget. It is understood that an oligonucleotide need not be 100%complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e. under physiological conditions in thecase of in vivo assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed.

[0048] Cleavage of oligonucleotides by nucleolytic enzymes require theformation of an enzyme-substrate complex, or in particular anuclease-oligonucleotide complex. The nuclease enzymes will generallyrequire specific binding sites located on the oligonucleotides forappropriate attachment. If the oligonucleotide binding sites are removedor blocked, such that nucleases are unable to attach to theoligonucleotides, the oligonucleotides will be nuclease resistant. Inthe case of restriction endonucleases that cleave sequence-specificpalindromic double-stranded DNA, certain binding sites such as the ringnitrogen in the 3- and 7-positions have been identified as requiredbinding sites. Removal of one or more of these sites or stericallyblocking approach of the nuclease to these particular positions withinthe oligonucleotide has provided various levels of resistance tospecific nucleases.

[0049] This invention provides oligonucleotides possessing superiorhybridization properties. Structure-activity relationship studies haverevealed that an increase in binding (T_(m)) of certain 2′-sugarmodified oligonucleotides to an RNA target (complement) correlates withan increased “A” type conformation of the heteroduplex. Furthermore,absolute fidelity of the modified oligonucleotides is maintained.Increased binding of 2′-sugar modified sequence-specificoligonucleotides of the invention provides superior potency andspecificity compared to phosphorus-modified oligonucleotides such asmethyl phosphonates, phosphate triesters and phosphoramidates as knownin the literature.

[0050] The only structural difference between DNA and RNA duplexes is ahydrogen atom at the 2′-position of the sugar moiety of a DNA moleculeversus a hydroxyl group at the 2′-position of the sugar moiety of an RNAmolecule (assuming that the presence or absence of a methyl group in theuracil ring system has no effect). However, gross conformationaldifferences exist between DNA and RNA duplexes.

[0051] It is known from X-ray diffraction analysis of nucleic acidfibers [Arnott and Hukins, Biochemical and Biophysical ResearchCommunication, 47, 1504-1510 (1970)] and analysis of crystals ofdouble-stranded nucleic acids that DNA takes a “B” form structure andRNA takes the more rigid “A” form structure. The difference between thesugar puckering (C2′ endo for “B” form DNA and C3′ endo for “A” formRNA) of the nucleosides of DNA and RNA is the major conformationaldifference between double-stranded nucleic acids.

[0052] The primary contributor to the conformation of the pentofuranosylmoiety is the nature of the substituent at the 2′-position. Thus, thepopulation of the C3′-endo form increases with respect to the C2′-endoform as the electronegativity of the 2′-substituent increases. Forexample, among 2′-deoxy-2′-haloadenosines, the 2′-fluoro derivativeexhibits the largest population (65%) of the C3′-endo form, and the2′-iodo exhibits the lowest population (7%). Those of adenosine (2′-OH)and deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore,the effect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoroadenosine) is furthercorrelated to the stabilization of the stacked conformation. Researchindicates that dinucleoside phosphates have a stacked conformation witha geometry similar to that of A-A but with a greater extent of base-baseoverlapping than A-A. It is assumed that the highly polar nature of theC2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an “A” structure.

[0053] Data from UV hypochromicity, circular dichromism, and ¹H NMR alsoindicate that the degree of stacking decreases as the electronegativityof the halo substituent decreases. Furthermore, steric bulk at the2′-position of the sugar moiety is better accommodated in an “A” formduplex than a “B” form duplex.

[0054] Thus, a 2′-substituent on the 3′-nucleotidyl unit of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent.

[0055] The 2′-iodo substituted nucleosides possess the lowest C3′-endopopulation (7%) of the halogen series. Thus, based solely on stericeffects, one would predict that a 2′-iodo (or other similar group) wouldcontribute stacking destabilization properties, and thus reduced binding(T_(m)) of the oligonucleotides. However, the lower electronegativityand high hydrophobicity of the iodine atom (or another similar group)complicates the ability to predict stacking stabilities and bindingstrengths.

[0056] Studies with a 2′-OMe modification of 2′-deoxy guanosine,cytidine, and uridine dinucleoside phosphates exhibit enhanced stackingeffects with respect to the corresponding unmethylated species (2′-OH).In this case, the hydrophobic attractive forces of the methyl group tendto overcome the destablilizing effects of its steric bulk.

[0057] 2′-Fluoro-2′-deoxyadenosine has been determined to have anunusually high population of 3′-endo puckered form among nucleosides.Adenosine, 2′-deoxyadenosine and other derivatives have less than 40% oftheir population in the 3′-endo conformation. It is known that anucleoside residue in well-stacked oligonucleotides favors 3′-endoribofuranose puckering.

[0058] Melting temperatures (complementary binding) are increased withthe 2′-substituted adenosine diphosphates. It is not clear whether the3′-endo preference of the conformation or the presence of thesubstituent is responsible for the increased binding. However, greateroverlap of adjacent bases (stacking) can be achieved with the 3′-endoconformation.

[0059] Compounds of the invention can be utilized as diagnostics,therapeutics and as research reagents and kits. They can be utilized inpharmaceutical compositions by adding an effective amount of anoligonucleotide of the invention to a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism can be contacted with an oligonucleotide of theinvention having a sequence that is capable of specifically hybridizingwith a strand of target nucleic acid that codes for the undesirableprotein.

[0060] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligomer in accordance with the invention, commonly in apharmaceutically acceptable carrier, in doses ranging from 0.01 μg to100 g per kg of body weight depending on the age of the patient and theseverity of the disease state being treated. Further, the treatment maybe a single dose or may be a regimen that may last for a period of timewhich will vary depending upon the nature of the particular disease, itsseverity and the overall condition of the patient, and may extend fromonce daily to once every 20 years. Following treatment, the patient ismonitored for changes in his/her condition and for alleviation of thesymptoms of the disease state. The dosage of the oligomer may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, or if the disease statehas been ablated.

[0061] In some cases it may be more effective to treat a patient with anoligomer of the invention in conjunction with other traditionaltherapeutic modalities. For example, a patient being treated for AIDSmay be administered an oligomer in conjunction with AZT, or a patientwith atherosclerosis may be treated with an oligomer of the inventionfollowing angioplasty to prevent reocclusion of the treated arteries.

[0062] Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligomers, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to several years.

[0063] Following successful treatment, it may be desirable to have thepatient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligomer is administered in maintenancedoses, ranging from 0.01 μg to 100 g per kg of body weight, once or moredaily, to once every several years.

[0064] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

[0065] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0066] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0067] Compositions for intrathecal or intraventricular administrationmay include sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

[0068] Formulations for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives.

[0069] The present invention can be practiced in a variety of organismsranging from unicellular prokaryotic and eukaryotic organisms tomulticellular eukaryotic organisms. Any organism that utilizes DNA-RNAtranscription or RNA-protein translation as a fundamental part of itshereditary, metabolic or cellular machinery is susceptible to suchtherapeutic and/or prophylactic treatment. Seemingly diverse organismssuch as bacteria, yeast, protozoa, algae, plant and higher animal forms,including warm-blooded animals, can be treated in this manner. Further,since each of the cells of multicellular eukaryotes also includes bothDNA-RNA transcription and RNA-protein translation as an integral part oftheir cellular activity, such therapeutics and/or diagnostics can alsobe practiced on such cellular populations. Furthermore, many of theorganelles, e.g. mitochondria and chloroplasts, of eukaryotic cells alsoinclude transcription and translation mechanisms. As such, single cells,cellular populations or organelles also can be included within thedefinition of organisms that are capable of being treated with thetherapeutic or diagnostic oligonucleotides of the invention. As usedherein, therapeutics is meant to include both the eradication of adisease state, killing of an organism, e.g. bacterial, protozoan orother infection, or control of aberrant or undesirable cellular growthor expression.

[0070] The present novel approach to obtaining stronger binding is toprepare RNA mimics that bind to target RNA. Therefore, a randomstructure-activity relationship approach was undertaken to discovernuclease resistant oligonucleotides that maintain appropriatehybridization properties.

[0071] A series of 2′-deoxy-2′-modified nucleosides of adenine, guanine,cytosine, thymidine and certain analogs of these nucleobases have beenprepared and incorporated into oligonucleotides via solid phase nucleicacid synthesis. These novel oligonucleotides were assayed for theirhybridization properties and their ability to resist degradation bynucleases compared to the unmodified oligonucleotides. Initially, smallelectronegative atoms or groups were selected because they would not beexpected to sterically interfere with required Watson-Crick base pairhydrogen bonding (hybridization). However, electronic changes due to theelectronegativity of the atom or group in the 2′-position may profoundlyaffect the sugar conformation. Structure-activity relationship studiesrevealed that the sugar-modified oligonucleotides hybridized to thetarget RNA more strongly than the unmodified 2′-deoxy oligonucleotides.

[0072] 2′-Substituted oligonucleotides were synthesized by standardsolid phase nucleic acid synthesis using an automated synthesizer suchas Model 380B (Perkin-Elmer/Applied Biosystems) or MilliGen/Biosearch7500 or 8800. Triester, phosphoramidite, or hydrogen phosphonatecoupling chemistries [Oligonucleotides. Antisense Inhibitors of GeneExpression. M. Caruthers, p. 7, J. S. Cohen (Ed.), CRC Press, BocaRaton, Fla., 1989] are used with these synthesizers to provide thedesired oligonucleotides. The Beaucage reagent [J. Amer. Chem. Soc.,112, 1253 (1990)] or elemental sulfur [Beaucage et al., Tet. Lett., 22,1859 (1981)] is used with phosphoramidite or hydrogen phosphonatechemistries to provide 2′-substituted phosphorothioate oligonucleotides.

[0073] The requisite 2′-substituted nucleosides (A, G, C, T(U), andother modified nucleobases) were prepared by modification of severalliterature procedures as described below.

[0074] Procedure 1. Nucleophilic Displacement of 2′-Leaving Group inArabino Purine Nucleosides. Nucleophilic displacement of a leaving groupin the 2′-up position (2′-deoxy-2′-(leaving group)arabino sugar) ofadenine or guanine or their analog nucleosides. General syntheticprocedures of this type have been described by Ikehara et al.,Tetrahedron, 34, 1133 (1978); ibid., 31, 1369 (1975); Chemistry andPharmaceutical Bulletin, 26, 2449 (1978); ibid., 26, 240 (1978);Ikehara, Accounts of Chemical Research, 2, 47 (1969); and Ranganathan,Tetrahedron Letters, 15, 1291 (1977).

[0075] Procedure 2. Nucleophilic Displacement of 2,2′-AnhydroPyrimidines. Nucleosides thymine, uracil, cytosine or their analogs areconverted to 2′-substituted nucleosides by the intermediacy of2,2′-cycloanhydro nucleoside as described by Fox et al., Journal ofOrganic Chemistry, 29, 558 (1964).

[0076] Procedure 3. 2′-Coupling Reactions. Appropriately 3′,5′-sugar andbase protected purine and pyrimidine nucleosides having a unprotected2′-hydroxyl group are coupled with electrophilic reagents such as methyliodide and diazomethane to provide the mixed sequences containing a2′-OMe group H. Inoue et al., Nucleic Acids Research, 15, 6131.

[0077] Procedure 4. 2-Deoxy-2-substituted Ribosylations.2-Substituted-2-deoxyribosylation of the appropriately protected nucleicacid bases and nucleic acids base analogs has been reported by Jarvi etal., Nucleosides & Nucleotides, 8, 1111-1114 (1989) and Hertel et al.,Journal of Organic Chemistry, 53, 2406 (1988).

[0078] Procedure 5. Enzymatic Synthesis of 2′-Deoxy-2′-SubstitutedNucleosides. The 2-Deoxy-2-substituted glycosyl transfer from onenucleoside to another with the aid of pyrimidine and purine ribo ordeoxyribo phosphorolyses has been described by Rideout and Krenitsky,U.S. Pat. No. 4,381,344 (1983).

[0079] Procedure 6. Conversion of 2′-Substituents Into New Substituents.2′-Substituted-2′-deoxynucleosides are converted into new substituentsvia standard chemical manipulations. For example, Chladek et al.[Journal of Carbohydrates, Nucleosides & Nucleotides, 7, 63 (1980)]describes the conversion of 2′-deoxy-2′-azidoadenosine, prepared fromarabinofuranosyladenine, into 2′-deoxy-2′-aminoadenosine.

[0080] Procedure 7. Free Radical Reactions. Conversions of halogensubstituted nucleosides into 2′-deoxy-2′-substituted nucleosides viafree radical reactions has been described by Parkes and Taylor[Tetrahedron Letters, 29, 2995 (1988)].

[0081] Procedure 8. Conversion of Ribonucleosides to2′-Deoxy-2′-Substituted Nucleoside. Appropriately 3′,5′-sugar and baseprotected purine and pyrimidine nucleosides having a unprotected2′-hydroxyl group are converted to 2′-deoxy-2′-substituted nucleosidesby the process of oxidation to the 2′-keto group, reaction withnucleophilic reagents , and finally 2′-deoxygenation. Procedures of thistype have been described by De las Heras, et al. [Tetrahedron Letters,29, 941 (1988)].

[0082] Procedure 9. In one process of the invention, 2′-deoxysubstituted guanosine compounds are prepared via an(arabinofuranosyl)guanine intermediate obtained via anoxidation-reduction reaction. A leaving group at the 2′ position of thearabinofuranosyl sugar moiety of the intermediate arabino compound isdisplaced via an SN₂ reaction with an appropriate nucleophile. Thisprocedure thus incorporates principles of both Procedure 1 and Procedure8 above. 2′-Deoxy-2′-fluoroguanosine is preferably prepared via thisprocedure. The intermediate arabino compound was obtained utilizing avariation of the oxidation-reduction procedure of Hansske et al.[Tetrahedron, 40, 125 (1984)]. According to this invention, thereduction was effected starting at −78° C. and allowing the reductionreaction to exothermically warm to about −2° C. This results in a highyield of the intermediate arabino compound.

[0083] In conjunction with use of a low temperature reduction,utilization of a tetraisopropyldisiloxane blocking group (a “TPDS”group) for the 3′ and 5′ positions of the starting guanosine compoundcontributes to an improved ratio of intermediate arabino compound to theribo compound following oxidation and reduction. Following oxidation andreduction, the N² guanine amino nitrogen and the 2′-hydroxyl moieties ofthe intermediate arabino compound are blocked with isobutyryl protectinggroups (“Ibu” groups). The tetraisopropyldisiloxane blocking group isremoved and the 3′ and 5′ hydroxy groups are further protected with asecond blocking group, a tetrahydropyranyl blocking group (“THP” group).The isobutyryl group is selectively removed from 2′-hydroxyl groupfollowed by derivation of the 2′ position with a triflate leaving group.The triflate group was then displaced with inversion about the 2′position to yield the desired 2′-deoxy-2′-fluoroguanosine compound.

[0084] In addition to the triflate leaving group, other leaving groupsinclude, but are not limited to, alkylsulfonyl, substitutedalkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,heterocyclosulfonyl or trichloroacetimidate. Representative examplesinclude p-(2,4-dinitroanilino)-benzenesulfonyl, benzenesulfonyl,methylsulfonyl, p-methyl-benzenesulfonyl, p-bromobenzenesulfonyl,trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl and 2,4,6-trichlorophenyl.

[0085] The isobutyryl group remaining on the N² heterocyclic aminomoiety of the guanine ring can be removed to yield a completelydeblocked nucleoside. However, preferably, for incorporation of the2′-deoxy-2′-substituted compound into an oligonucleotide, deblocking ofthe 2 isobutyryl protecting group is deferred until afteroligonucleotide synthesis is complete. Normally for use in automatednucleic acid synthesizers, blocking of the N² guanine moiety with anisobutyryl group is preferred. Thus, advantageously, theN²-isobutyryl-blocked 2′-deoxy-2′-substituted guanosine compoundsresulting from the method of the invention can be directly used foroligonucleotide synthesis on automated nucleic acid synthesizers.

[0086] For the purpose of illustration, the oligonucleotides of theinvention have been used in a ras-luciferase fusion system usingras-luciferase transactivation. As described in InternationalPublication Number WO 92/22651, published Dec. 23, 1992 and commonlyassigned with this application, the entire contents of which are hereinincorporated by reference, the ras oncogenes are members of a genefamily that encode related proteins that are localized to the inner faceof the plasma membrane. Ras proteins have been shown to be highlyconserved at the amino acid level, to bind GTP with high affinity andspecificity, and to possess GTPase activity. Although the cellularfunction of ras gene products is unknown, their biochemical properties,along with their significant sequence homology with a class ofsignal-transducing proteins known as GTP binding proteins, or Gproteins, suggest that ras gene products play a fundamental role inbasic cellular regulatory functions relating to the transduction ofextracellular signals across plasma membranes.

[0087] Three ras genes, designated H-ras, K-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The most commonly detectedactivating ras mutation found in human tumors is in codon-12 of theH-ras gene in which a base change from GGC to GTC results in aglycine-to-valine substitution in the GTPase regulatory domain of theras protein product. This single amino acid change is thought to abolishnormal control of ras protein function, thereby converting a normallyregulated cell protein to one that is continuously active. It isbelieved that such deregulation of normal ras protein function isresponsible for the transformation from normal to malignant growth.

[0088] The oligonucleotides of the present invention have also been usedfor modulating the expression of the raf gene, a naturally presentcellular gene which occasionally converts to an activated form that hasbeen implicated in abnormal cell proliferation and tumor formation.

[0089] The oligonucleotides of the present invention are alsospecifically hybridizable with nucleic acids relating to protein kinaseC (PKC). These oligonucleotides have been found to modulate theexpression of PKC.

[0090] The following examples illustrate the present invention and arenot intended to limit the same.

EXAMPLE 1 Preparation of 2′-Deoxy-2′-fluoro Modified Oligonucleotides

[0091] A.N⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-di-methoxytrityl)]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite.

[0092] N⁶-Benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine was preparedfrom 9-β-D-arabinofuranosyladenine in a five-step synthesis using amodification of a procedure reported by Ikehara at al. [Nucleosides andNucleotides, 2, 373-385 (1983)]. The N⁶-benzoyl derivative was obtainedin good yield utilizing the method of transient protection withchlorotrimethylsilane. Jones [J. Am. Chem. Soc., 104, 1316 (1982)].Selective protection of the 3′ and 5′-hydroxyl groups ofN⁶-Benzoyl-9-β-D-arabinofuranosyladenine with tetrahydropyranyl (THP)was accomplished by modification of the literature procedure accordingto Butke et al. [Nucleic Acid Chemistry, Part 3, p.149, L. B. Townsendand R. S. Tipson, Eds., J. Wiley and Sons, New York, 1986], to yieldN⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabino-furanosyl]adeninein good yield. Treatment ofN⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabino-furanosyl]adeninewith trifluoromethanesulfonic anhydride in dichloromethane gave the2′-triflate derivativeN⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninewhich was not isolated due to its lability. Displacement of the2′-triflate group was effected by reaction with tetrabutylammoniumfluoride in tetrahydrofuran to obtain a moderate yield of the 2′-fluoroderivative N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine. Deprotection of the THP groups of N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl]adeninewas accomplished by treatment with Dowex-50W in methanol to yieldN⁶-benzoyl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine in moderateyield. The ¹H-NMR spectrum was in agreement with the literature values.[Ikehara and Miki, Chem. Pharm. Bull., 26, 2449 (1978)]. Standardmethodologies were employed to obtain the5′-dimethoxytrityl-3′-phosphoramidite intermediatesN⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenineandN⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite.[Ogilvie, Can J. Chem., 67, 831-839 (1989)].

[0093] B. N⁶-Benzoyl-9-β-D-arabinofuranosyladenine.

[0094] 9-β-D-arabinofuranosyladenine (1.07 g, 4.00 mmol) was dissolvedin anhydrous pyridine (20 mL) and anhydrous dimethylformamide (20 mL)under an argon atmosphere. The solution was cooled to 0° C. andchlorotrimethylsilane (3.88 mL, 30.6 mmol) was added slowly to thereaction mixture via a syringe. After stirring the reaction mixture at0° C. for 30 minutes, benzoyl chloride (2.32 mL, 20 mmol) was addedslowly. The reaction mixture was allowed to warm to 20° C. and stirredfor 2 hours. After cooling the reaction mixture to 0° C., cold water (8mL) was added and the mixture was stirred for 15 minutes. Concentratedammonium hydroxide (8 mL) was slowly added to the reaction mixture togive a final concentration of 2 M of ammonia. After stirring the coldreaction mixture for 30 minutes, the solvent was evaporated in vacuo (60torr) at 20° C. followed by evaporation in vacuo (1 torr) at 40° C. togive an oil. This oil was triturated with diethyl ether (50 mL) to givea solid which was filtered and washed with diethyl ether three times.This crude solid was triturated in methanol (100 mL) at refluxtemperature three times and the solvent was evaporated to yieldN⁶-Benzoyl-9-β-D-arabino-furanosyladenine as a solid (1.50 g, 100%).

[0095] C.N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine.

[0096] N⁶-Benzoyl-9-β-D-arabinofuranosyladenine (2.62 g, 7.06 mmol) wasdissolved in anhydrous dimethylformamide (150 mL) under argon andp-toluenesulfonic acid monohydrate (1.32 g, 6.92 mmol) was added. Thissolution was cooled to 0° C. and dihydropyran (1.26 mL, 13.8 mmol) wasadded via a syringe. The reaction mixture was allowed to warm to 20° C.Over a period of 5 hours a total of 10 equivalents of dihydropyran wereadded in 2 equivalent amounts in the fashion described. The reactionmixture was cooled to 0° C. and saturated aqueous sodium bicarbonate wasadded slowly to a pH of 8, then water was added to a volume of 750 mL.The aqueous mixture was extracted with methylene chloride (4×200 mL),and the organic phases were combined and dried over magnesium sulfate.The solids were filtered and the solvent was evaporated in vacuo (60torr) at 30° C. to give a small volume of liquid which was evaporated invacuo (1 torr) at 40° C. to give an oil. This oil was coevaporated withp-xylene in vacuo at 40° C. to give an oil which was dissolved inmethylene chloride (100 mL). Hexane (200 mL) was added to the solutionand the lower-boiling solvent was evaporated in vacuo at 30° C. to leavea white solid suspended in hexane. This solid was filtered and washedwith hexane (3×10 mL) then purified by column chromatography usingsilica gel and methylene chloride-methanol (93:7) as the eluent. Thefirst fraction yielded the title compound 3 as a white foam (3.19 g,83%) and a second fraction gave a white foam (0.81 g) which wascharacterized as the 5′-monotetrahydropyranyl derivative ofN⁶-Benzoyl-9-β-D-arabinofuranosyladenine.

[0097] D.N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine.

[0098]N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(2.65 g, 4.91 mmol) was dissolved in anhydrous pyridine (20 mL) and thesolvent was evaporated in vacuo (1 mm Hg) at 40° C. The resulting oilwas dissolved in anhydrous methylene chloride (130 mL) under argonanhydrous pyridine (3.34 mL, 41.3 mmol) and N,N-dimethylaminopyridine(1.95 g, 16.0 mmol) were added. The reaction mixture was cooled to 0° C.and trifluoromethanesulfonic anhydride (1.36 mL, 8.05 mmol) was addedslowly via a syringe. After stirring the reaction mixture at 0° C. for 1hour, it was poured into cold saturated aqueous sodium bicarbonate (140mL). The mixture was shaken and the organic phase was separated and keptat 0° C. The aqueous phase was extracted with methylene chloride (2×140mL). The organic extracts which were diligently kept cold were combinedand dried over magnesium sulfate. The solvent was evaporated in vacuo(60 torr) at 20° C. then evaporated in vacuo (1 torr) at 20° C. to giveN⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenineas a crude oil which was not purified further.

[0099] E.N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl]adenine.

[0100]N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(4.9 mmol) as a crude oil was dissolved in anhydrous tetrahydrofuran(120 mL) and this solution was cooled to 0° C. under argon.Tetrabutylammonium fluoride as the hydrate (12.8 g, 49.1 mmol) wasdissolved in anhydrous tetrahydrofuran (50 mL) and half of this volumewas slowly added via a syringe to the cold reaction mixture. Afterstirring at 0° C. for 1 hour, the remainder of the reagent was addedslowly. The reaction mixture was stirred at 0° C. for an additional 1hour, then the solvent was evaporated in vacuo (60 torr) at 20° C. togive an oil. This oil was dissolved in methylene chloride (250 mL) andwashed with brine three times. The organic phase was separated and driedover magnesium sulfate. The solids were filtered and the solvent wasevaporated to give an oil. The crude product was purified by columnchromatography using silica gel in a sintered-glass funnel and ethylacetate was used as the eluent.N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninewas obtained as an oil (2.03 g, 76%).

[0101] F. N⁶-Benzoyl-9-(2′-fluoro-β-D-ribofuranosyl)adenine.

[0102]N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(1.31 g, 2.42 mmol) was dissolved in methanol (60 mL), and Dowex50W×2-100 (4 cm³, 2.4 m.eq) was added to the reaction mixture. Thereaction mixture was stirred at 20° C. for 1 hour then cooled to 0° C.Triethylamine (5 mL) was then slowly added to the cold reaction mixtureto a pH of 12. The resin was filtered and washed with 30% triethylaminein methanol until the wash no longer contained UV absorbing material.Toluene (50 mL) was added to the washes and the solvent was evaporatedat 24° C. in vacuo (60 torr, then 1 torr) to give a residue. Thisresidue was partially dissolved in methylene chloride (30 mL) and thesolvent was transferred to a separatory funnel. The remainder of theresidue was dissolved in hot (60° C.) water and after cooling thesolvent it was also added to the separatory funnel. The biphasic systemwas extracted, and the organic phase was separated and extracted withwater (3×100 mL). The combined aqueous extracts were evaporated in vacuo(60 torr, then 1 torr Hg) at 40° C. to give an oil which was evaporatedwith anhydrous pyridine (50 mL). This oil was further dried in vacuo (1torr Hg) at 20° C. in the presence of phosphorous pentoxide overnight togive N⁶-benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine as a yellow foam(1.08 g, 100%) which contained minor impurities.

[0103] G.N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxy-trityl)-β-D-ribofuranosyl]adenine.

[0104] N⁶-Benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine (1.08 g, 2.89mmol) which contained minor impurities was dissolved in anhydrouspyridine (20 mL) under argon and dry triethylamine (0.52 mL, 3.76 mmol)was added followed by addition of 4,4′-dimethoxytrityl chloride (1.13 g,3.32 mmol). After 4 hours of stirring at 20° C. the reaction mixture wastransferred to a separatory funnel and diethyl ether (40 mL) was addedto give a white suspension. This mixture was washed with water threetimes (3×10 ml), the organic phase was separated and dried overmagnesium sulfate. Triethylamine (1 ml) was added to the solution andthe solvent was evaporated in vacuo (60 torr Hg) at 20° C. to give anoil which was evaporated with toluene (20 mL) containing triethylamine(1 mL). This crude product was purified by column chromatography usingsilica gel and ethyl acetate-triethylamine (99:1) followed by ethylacetate-methanol-triethylamine (80:19:1) to give the product in twofractions. The fractions were evaporated in vacuo (60 torr, then 1 torrHg) at 20° C. to give a foam which was further dried in vacuo (1 torrHg) at 20° C. in the presence of sodium hydroxide to giveN⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenineas a foam (1.02 g, 52%).

[0105] H.N⁶-Benzoyl-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl]adenosine-3′-O-N,N-diisopropyl-β-cyanoethylphosphoramidite.

[0106]N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenine(1.26 g, 1.89 mmol) was dissolved in anhydrous dichloromethane (13 mL)under argon, diisopropylethylamine (0.82 mL, 4.66 mmol) was added, andthe reaction mixture was cooled to 0° C. Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 mL, 4.03 mmol) was addedto the reaction mixture which was allowed to warm to 20° C. and stirredfor 3 hours. Ethylacetate (80 mL) and triethylamine (1 mL) were addedand this solution was washed with brine (3×25 mL). The organic phase wasseparated and dried over magnesium sulfate. After filtration of thesolids the solvent was evaporated in vacuo at 20° C. to give an oilwhich was purified by column chromatography using silica gel andhexanes-ethyl acetate-triethyl-amine (50:49:1) as the eluent.Evaporation of the fractions in vacuo at 20° C. gave a foam which wasevaporated with anhydrous pyridine (20 mL) in vacuo (1 torr) at 26° C.and further dried in vacuo (1 torr Hg) at 20° C. in the presence ofsodium hydroxide for 24 h to give N⁶-Benzoyl-[2′-deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramiditeas a foam (1.05 g, 63%).

[0107] I.2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)uridine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0108] 2,2′-Cyclouridine is treated with a solution of 70% hydrogenfluoride/pyridine in dioxane at 120° C. for ten hours to provide aftersolvent removal a 75% yield of 2′-deoxy-2′-fluorouridine. The 5′-DMT and3′-cyanoethoxydiisopropyl-phosphoramidite derivitized nucleoside isobtained by standard literature procedures [Gait, Ed., OligonucleotideSynthesis. A Practical Approach, IRL Press, Washington, D.C. (1984)], oraccording to the procedure of Example 1A.

[0109] J.2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0110] 2′-Deoxy-2′-fluorouridine (2.51 g, 10.3 mmol) was converted tocorresponding cytidine analog via the method of C. B. Reese, et al., J.Chem. Soc. Perkin Trans I, pp. 1171-1176 (1982), by acetylation withacetic anhydride (3.1 mL, 32.7 mmol) in anhydrous pyridine (26 mL) atroom temperature. The reaction was quenched with methanol, the solventwas evaporated in vacuo (1 torr) to give an oil which was coevaporatedwith ethanol and toluene. 3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wascrystallized from ethanol to afford colorless crystals (2.38 g, 81%).

[0111]N-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained in a 70% yield (2.37 g) by reaction of3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine (2.75 g, 9.61 mmol) with1,2,4-triazole (5.97 g, 86.5 mmol), phosphorus oxychloride (1.73 mL,18.4 mmol), and triethylamine (11.5 mL, 82.7 mmol) in anhydrousacetonitrile at room temperature. After 90 min the reaction mixture wascooled to ice temperature and triethylamine (7.98 ml, 56.9 mmol) wasadded followed by addition of water (4.0 ml). The solvent was evaporatedin vacuo (1 torr) to give an oil which was dissolved in methylenechloride and washed with saturated aqueous sodium bicarbonate. Theaqueous phase was extracted with methylene chloride twice (2×100 mL) andthe organic extracts dried with magnesium sulfate. Evaporation of thesolvent afforded an oil from which the productN-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained by crystallization from ethanol.

[0112] 2′-deoxy-2′-fluorocytidine was afforded by treatment of protectedtriazol-1-yl derivative with concentrated ammonium hydroxide (4.26 mL,81.2 mmol) in dioxane at room temperature for 6 hours. After evaporationof the solvent the oil was stirred in half-saturated (at icetemperature) ammonia in methanol for 16 hours. The solvent wasevaporated and 2′-deoxy-2′-fluoro-cytidine crystallized fromethyl-acetate-methanol (v/v, 75:25) to give colorless crystals (1.24 g,75%).

[0113] N-4-benzoyl-2′-deoxy-2′-fluorocytidine was prepared by selectivebenzoylation with benzoic anhydride in anhydrous dimethylformamide, V.Bhat, et al. Nucleosides Nucleotides, Vol. 8, pp. 179-183 (1989). The5′-O-(4,4′-dimethoxytrityl)-3′-O-(N,N-diisopropyl-β-cyanoethyl-phosphoramidite)was prepared in accordance with Example 1A.

[0114] K.9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine.

[0115] The 3′ and 5′ positions of guanosine were protected by theaddition of a TPDS (1,1,3,3-tetraisopropyldisilox-1,3-diyl) protectinggroup as per the procedure of Robins et al. [Can. J. Chem., 61, 1911(1983)]. To a stirred solution of DMSO (160 mL) and acetic anhydride (20mL) was added the TPDS guanosine (21 g, 0.040 mol). The reaction wasstirred at room temperature for 36 hours and then cooled to 0° C. Coldethanol (400 mL, 95%) was added and the reaction mixture further cooledto −78° C. in a dry ice/acetone bath. NaBH₄ (2.0 g, 1.32 mol. eq.) wasadded. The reaction mixture was allowed to warm up to −2° C., stirredfor 30 minutes and again cooled to −78° C. This was repeated twice.After the addition of NaBH₄ was complete, the reaction was stirred at 0°C. for 30 minutes and then at room temperature for 1 hour. The reactionwas taken up in ethyl acetate (1 L) and washed twice with a saturatedsolution of NaCl. The organic layer was dried over MgSO₄ and evaporatedunder reduced pressure. The residue was coevaporated twice with tolueneand purified by silica gel chromatography using CH₂Cl₂—MeOH (9:1) as theeluent. Pure product (6.02 g) precipitated from the appropriate columnfractions during evaporation of these fractions, and an additional 11.49g of product was obtained as a residue upon evaporation of thefractions.

[0116] L.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine.

[0117]9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]β-D-arabinofuranosyl)guanine(6.5 g, 0.01248 mol) was dissolved in anhydrous pyridine (156 mL) underargon. DMAP (9.15 g) was added. Isobutyric anhydride (6.12 mL) wasslowly added and the reaction mixture stirred at room temperatureovernight. The reaction mixture was poured into cold saturated NaHCO₃(156 mL) and stirred for 10 minutes. The aqueous solution was extractedthree times with ethyl acetate (156 mL). The organic phase was washedthree times with saturated NaHCO₃ and evaporated to dryness. The residuewas coevaporated with toluene and purified by silica gel columnchromatography using CH₂Cl₂-acetone (85:15) to yield 5.67 g of product.

[0118] M. N²-Isobutyryl-9-(2′-O-isobutyryl-β-D-arabinofuranosyl)guanine.

[0119]N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine(9.83 g, 0.01476 mol) was dissolved in anhydrous THF (87.4 mL) at roomtemperature under argon. 1 M (nBu)₄N⁺F⁻ in THF (29.52 mL, 2 eq.) wasadded and the reaction mixture stirred for 30 minutes. The reactionmixture was evaporated at room temperature and the residue purified bysilica gel column chromatography using EtOAc—MeOH (85:15) to yield 4.98g (80%) of product.

[0120] N.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine.

[0121] N² -Isobutyryl-9-(2′-isobutyryl-β-D-arabinofuranosyl)guanine (4.9g) was dissolved in anhydrous 1,4-dioxane (98 mL) at room temperatureunder argon. p-Toluenesulphonic acid monohydrate (0.97 g) was addedfollowed by 3,4-dihydro-2H-pyran (DHP, 9.34 mL, 8.8 eq.). The reactionmixture was stirred for 2 hours, then cooled to 0° C. and saturatedNaHCO₃ (125 mL) was added to quench the reaction. The reaction mixturewas extracted three times with 125 mL portions of CH₂Cl₂ and the organicphase dried over MgSO₄. The organic phase was evaporated and the residuedissolved in minimum volume of CH₂Cl₂, but in an amount sufficient toyield a clear liquid not a syrup, and then dripped into hexane (100times the volume of CH₂Cl₂). The precipitate was filtered to yield 5.59(81.5%) of product.

[0122] O.N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine.

[0123] N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine (5.58 g) was dissolved in pyridine-MeOH—H₂O (65:30:15, 52 mL) atroom temperature. The solution was cooled to 0° C. and 52 mL of 2 N NaOHin EtOH—MeOH (95:5) was added slowly, followed by stirring for 2 hoursat 0° C. Glacial acetic acid was added to pH 6, and saturated NaHCO₃ wasadded to pH 7. The reaction mixture was evaporated under reducedpressure and the residue coevaporated with toluene. The residue was thendissolved in EtOAc (150 mL) and washed 3× with saturated NaHCO₃. Theorganic phase was evaporated and the residue purified by silica gelcolumn chromatography using EtOAc—MeOH (95:5) as the eluent, yielding3.85 g (78.3%) of product.

[0124] P. N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guanine.

[0125]N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine(3.84 g) was dissolved in anhydrous CH₂Cl₂ (79 mL), anhydrous pyridine(5 mL) and DMAP (2.93 g) at room temperature under argon. The solutionwas cooled to 0° C. and trifluoromethanesulfonic anhydride (1.99 mL) wasslowly added with stirring. The reaction mixture was stirred at roomtemperature for 1 hour then poured into 100 mL of saturated NaHCO₃. Theaqueous phase was extracted three times with cold CH₂Cl₂. The organicphase was dried over MgSO₄, evaporated and coevaporated with anhydrousMeCN to yield a crude product.

[0126] Q.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-ribofuranosyl)guanine.

[0127] The crude product from Example 1-P, i.e.N²-isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guaninewas dissolved in anhydrous THF (113 mL) under argon at 0° C. 1 M(nBu)₄N⁺F⁻ (dried by coevaporation with pyridine) in THF (36.95 mL) wasadded with stirring. After 1 hour, a further aliquot of (nBu)₄N⁺F⁻ inTHF (36.95 mL) was added. The reaction mixture was stirred at 0° C. for5 hours and stored overnight at −30° C. The reaction mixture wasevaporated under reduced pressure and the residue dissolved in CH₂Cl₂(160 mL) and extracted five times with deionized water. The organicphase was dried over MgSO₄ and evaporated. The residue was purified bysilica gel column chromatography using EtOAc—MeOH (95:5) to yield 5.25 gof product.

[0128] R. N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine.

[0129]N²-isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-ribofuranosyl)guanine(3.85 g) was dissolved in MeOH (80 mL) at room temperature. Pre-washedDowex 50W resin (12.32 cm³) was added and the reaction mixture stirredat room temperature for 1 hour. The resin was filtered and the filtrateevaporated to dryness. The resin was washed withpyridine-triethylamine-H₂O (1:3:3) until filtrate was clear. Thisfiltrate was evaporated to obtain an oil. The residues from bothfiltrates were combined in H₂O (200 mL) and washed with CH₂Cl₂ (3×100mL). The aqueous phase was evaporated to dryness and the residuerecrystallized from hot MeOH to yield 0.299 g of product as a whitepowder. The remaining MeOH solution was purified by silica gel columnchromatography to further yield 0.783 g of product by elution withEtOH—MeOH (4:1).

[0130] S.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxytrityl]-β-D-ribofuranosyl)guanine.

[0131] N² -isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine(1.09 g) was dissolved in pyridine (20 mL) and triethylamine (0.56 mL)at room temperature under argon. 4,4′-Dimethoxytrityl chloride (1.20 g,1.15 molar eq.) was added and the reaction mixture stirred at roomtemperature for 5 hours. The mixture was transferred to a separatoryfunnel and extracted with diethyl ether (100 mL). The organic phase waswashed with saturated NaHCO₃ (3×70 mL), and the aqueous phaseback-extracted three times with diethyl ether. The combined organicphases were dried over MgSO₄ and triethylamine (4 mL) was added tomaintain the solution at basic pH. The solvent was evaporated and theresidue purified by silica gel column chromatography usingEtOAc-triethylamine (100:1) and then EtOAc—MeOH-triethylamine (95:5:1)as eluents yielding 1.03 g of product.

[0132] T.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxytrityl]-guanosine-3′-O-N,N-diisopropyl-β-D-cyanoethylphosphoramidite.

[0133]N²-isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4′-dimethoxytrityl])-β-D-ribofuranosyl)guanine(0.587 g) was dissolved in anhydrous CH₂Cl₂ (31 mL) anddiisopropylethylamine (0.4 mL) at room temperature under argon. Thesolution was cooled to 0° C. andchloro(diisopropylamino)-β-cyanoethoxyphosphine (0.42 mL) was slowlyadded. The reaction mixture was allowed to warm to room temperature andstirred for 3.5 hours. CH₂Cl₂-triethylamine (100:1, 35 mL) was added andthe mixture washed with saturated NaHCO₃ (6 mL). The organic phase wasdried over MgSO₄ and evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usinghexane-EtOAc-triethylamine (75:25:1) for 2 column volumes, thenhexane-EtOAc-triethylamine (25:75:1), and finally EtOAc-triethylamine.The product-containing fractions were pooled and the solvent evaporatedunder reduced pressure. The resulting oil was coevaporated twice withMeCN and dried under reduced pressure. The resulting white solid wasdissolved in CH₂Cl₂ (3 mL) and dripped into stirring hexane (300 mL).The resulting precipitate was filtered and dried under reduced pressureto yield 0.673 g (88%) of product.

EXAMPLE 2 Preparation of 2′-Deoxy-2′-cyano Modified Oligonucleotides

[0134] A.N⁶-Benzoyl-[2′-deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0135] 2′-Deoxy-2′-cyanoadenosine is prepared by the free radicalreplacement of the 2′-iodo group of2′-deoxy-2′-iodo-3′,5′-O-(disiloxytetraisopropyl)-N⁶-benzoyladenosineaccording to a similar procedure described by Parkes and Taylor[Tetrahedron Letters, 29, 2995 (1988)]. 2′-Deoxy-2′-iodoadenosine wasprepared by Ranganathan as described in Tetrahedron Letters, 15, 1291(1977), and disilyated as described by Markiewicz and Wiewiorowski[Nucleic Acid Chemistry, Part 3, pp. 222-231, L. B. Townsend and R. S.Tipson, Eds., J. Wiley and Sons, New York, 1986. This material istreated with hexamethylditin, AIBN, and t-butylisocyanate in toluene toprovide protected 2′-deoxy-2′-cyanoadenosine. This material, afterselective deprotection, is converted to its 5′-DMT-3′-phosphoramidite asdescribed in Example 1A.

[0136] B.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-uridine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0137] 2′-Deoxy-2′-iodouridine (or 5-methyluridine), 3′,5′-disilylatedas described above, is converted to the 2′-iodo derivative bytriphenylphosphonium methyl iodide treatment as described by Parkes andTaylor [Tetrahedron Letters, 29, 2995 (1988)]. Application of freeradical reaction conditions as described by Parkes and Taylor providesthe 2′-cyano group of the protected nucleoside. Deprotection of thismaterial and subsequent conversion to the protected monomer as describedabove provides the requisite phosphoramidite.

[0138] C.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0139] 2′-Deoxy-2′-iodocytidine is obtained from the correspondinguridine compound described above via a conventional keto to aminoconversion.

[0140]D.2′-Deoxy-2′-cyano-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O-(N,N-diisopropyl-b-cyanoethylphosphoramidite).

[0141] 2′-Deoxy-2′-cyanoguanosine is obtained by the displacement of thetriflate group in the 2′-position (arabinosugar) of 3′,5′-disilylatedN²-isobutrylguanosine. Standard deprotection and subsequent reprotectionprovides the title monomer.

EXAMPLE 3 Preparation of 2′-Deoxy-2′-(trifluoromethyl) ModifiedOligonucleotides

[0142] The requisite 2′-deoxy-2′-trifluromethyribosides of nucleic acidbases A, G, U(T), and C are prepared by modifications of a literatureprocedure described by Chen and Wu [Journal of Chemical Society, PerkinTransactions, 2385 (1989)]. Standard procedures, as described in Example1A, are employed to prepare the 5′-DMT and 3′-phosphoramidites as listedbelow.

[0143] A.N⁶-Benzoyl-[2′-deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O-(N,N-di-isopropyl-β-cyanoethylphosphoramidite).

[0144] B.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)uridine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0145] C.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0146] D.2′-Deoxy-2′-trifluoromethyl-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 4 Preparation of 2′-Deoxy-2′-(trifluoromethoxy) ModifiedOligonucleotides

[0147] The requisite 2′-deoxy-2′-O-trifluromethyribosides of nucleicacid bases A, G, U(T), and C are prepared by modifications of literatureprocedures described by Sproat et al. [Nucleic Acids Research, 18, 41(1990)] and Inoue et al. [Nucleic Acids Research, 15:, 131 (1987)].Standard procedures, as described in Example 1A, are employed to preparethe 5′-DMT and 3′-phosphoramidites as listed below.

[0148] A.N⁶-Benzoyl-[2′-deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0149] B.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-uridine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0150] C.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0151] D.2′-Deoxy-2′-(trifluoromethoxy)-5′-O-(4,4′-dimethoxytrityl)-guanosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 5 Preparation of 2′-Deoxy-2′-O-alkyl Modified Oligonucleotides

[0152] Illustrative 2′-O-alkyl (2′-alkoxy) modified oligonucleotides areprepared from appropriate precursor nucleotides that in turn areprepared starting from a commercial nucleoside. The nucleoside, eitherunblocked or appropriately blocked as necessary to protected exocyclicfunctional groups on their heterobases, are alkylated at the 2′-Oposition. This 2′-O-alkylated nucleosides is converted to a5′-O-dimethoxytrityl protected nucleosides and 3′-O-phosphitylated togive a phosphoramidite. The phosphoramidites are incorporated inoligonucleotides using standard machine cycle solid phasephosphoramidite oligonucleotide chemistry. For illustrative purposes thesynthesis of 2-O-nonyladenosine, 2-O-propyluridine, 2-O-methylcytidine,2′-O-octadecylguanosine,2′-O-[(N-phthalimido)prop-3-yl]-N⁶-benzoyladenosine and2-O-[(imidazol-1-yl)but-4-yl]adenosine are given. Other 2′-O-alkylatednucleosides are prepared in a like manner using an appropriate startingalkyl halide in place of the illustrated alkyl halides. For certain2′-O-aminoalkyl compounds of the invention, protected amines, e.g.phthalimido, were used during alkylation, subsequent tritylation andphosphitylation. After incorporation into the oligonucleotide ofinterest, the 2′-O-protected aminoalkyl moiety are deblocked to yieldthe free amino compound, i.e 2′-O—(CH₂)_(n)—NH₂.

[0153] A.N⁶-Benzoyl-(2′-deoxy-2′-O-nonyl-5′-O-(4,4′-dimethoxytrityl]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0154] 2′-O-Nonyladenosine

[0155] To a solution of 10 g of adenosine in 400 ml of dimethylformamide was added 2.25 g of 60% sodium hydride (oil). After one hour,8.5 ml of 1-bromononane was added. The reaction was stirred for 16hours. Ice was added and the solution evaporated in vacuo. Water andethyl acetate were added. The organic phase was separated, dried, andevaporated in vacuo to give a white solid, which was recrystallized fromethanol to yield 4.8 g of the title compound, m.p. 143-144° C. analysisfor: C₁₉H₃₁N₅O₄. Calculated: C, 57.99; H, 7.94; N, 1779. Found: C,58.13; H, 7.93; N, 17.83.

[0156] 2′-O-Nonyl-N⁶-benzoyladenosine

[0157] 2′-O-Nonyladenosine was treated with benzoyl chloride in a mannersimilar to the procedure of B. L. Gaffney and R. A. Jones, TetrahedronLett., Vol. 23, p. 2257 (1982). After chromatography on silica gel(ethyl acetate-methanol), the title compound was obtained. Analysis for:C₂₆H₃₅N₅O₉. Calculated: C, 62.75; H, 7.09; N, 17.07. Found: C, 62.73; H,14.07; N, 13.87.

[0158] 2′-O-Nonyl-5′-O-dimethoxytrityl-N⁶-benzoyladenosine

[0159] To a solution of 4.0 g of 2′-O-nonyl-N⁶-benzoyladenosine in 250ml of pyridine was added 3.3 g of 4,4′-dimethoxytrityl chloride. Thereaction was stirred for 16 hours. The reaction was added toice/water/ethyl acetate, the organic layer was separated, dried, andconcentrated in vacuo to a gum. 5.8 g of the title compound was obtainedafter chromatography on silica gel (ethyl acetate-methanoltriethylamine). Analysis for: C₄₇H₅₃N₅O₇. Calculated: C, 70.56; H, 6.68;N, 8.75. Found: C, 70.26; H, 6.70; N, 8.71.

[0160]N⁶-Benzoyl-5′-O-dimethoxytrityl-2′-O-nonyladenosine-3′-O,N,N-diisopropyl-β-cyanoethylPhosphoramidite

[0161] 2′-O-nonyl-5′-O-dimethoxytrityl-N-benzoyladenosine was treatedwith (β-cyanoethoxy)chloro(N,N-diisopropyl)-aminephosphane in a mannersimilar to the procedure of F. Seela and A. Kehne, Biochemistry, Vol.26, p. 2233 (1987). After chromatography on silica gel (E=OAC/hexane),the title compound was obtained as a white foam.

[0162] B.2′-Deoxy-2′-O-propyl-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0163] 3′,5′-O-(1,1,3,3)Tetraisopropyl-1,3-disiloxanediyluridine

[0164] With stirring, uridine (40 g, 0.164 mol) and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol)were added to dry pyridine (250 mL). After stirring for 16 h at 25° C.,the reaction was concentrated under reduced pressure to an oil. The oilwas dissolved in methylene chloride (800 mL) and washed with sat'dsodium bicarbonate (200 g) scrub column. The product was recovered byelution with methylene chloride-methanol (97:3). The appropriatefractions were combined, evaporated under reduced pressure and dried at25° C./0.2 mmHg for 1 h to give 65 g (84%) of tan oil; TLC purity 95%(Rf 0.53, ethyl acetate-methanol 95:5); PMR (CDCl₃) δ 7.87 (d, 1, H-6),5.76 (d, 1, H-5), 5.81 (s, 1, H-1′).

[0165]N³-(4-Toluoyl)-3′-5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxane-diyluridine

[0166] 4-Toluoyl chloride (19.6 g, 0.127 mol) was added over 30 min to astirred solution of3′,5′-O-(1,1,3,3)-tetra-isopropyl-1,3-disiloxanediyluridine (56 g, 0.115mol) and triethylamine (15.1 g, 0.15 mol) in dimethylacetamide (400 mL)at 5° C. The mixture was allowed to warm to 25° C. for 3 h and thenpoured onto ice water (3.5 L) with stirring. The resulting solid wascollected, washed with ice water (3×500 mL) and dried at 45° C./0.2 mmHgfor 5 h to afford 49 g (70%) of tan solid; mp slowly softens above 45°C.; TLC purity ca. 95% (Rf 0.25, hexanes-ethyl acetate 4:1); PMR (DMSO)δ 7.9 (H-6), 7.9-7.4 (Bz), 5.8 (H-5), 5.65 (HO-2′), 5.6 (H-1′), 2.4(CH₃—Ar).

[0167]N³-(4-Toluoyl)-2′-O-propyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediyluridine

[0168] A mixture ofN³-(4-toluoyl)-3′-5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxane-diyluridine(88 g, 0.146 mol, 95% purity), silver oxide (88 g, 0.38 mol) and toluene(225 mL) was evaporated under reduced pressure. More toluene (350 mL)was added and an additional amount (100 mL) was evaporated. Under anitrogen atmosphere, propyl iodide was added in one portion and thereaction was stirred at 40° C. for 16 h. The silver salts were collectedand washed with ethyl acetate (3×150 mL). The combined filtrate wasconcentrated under reduced pressure. The residue was dissolved in aminimum of hexanes, applied on a silica gel column (800 g) and elutedwith hexanes-ethyl acetate (9:1→4:1). The appropriate fractions werecombined, concentrated under reduced pressure and dried at 25° C./0.2mmHg for 1 h to provide 68 g (74%) of tan oil; TLC purity 95% (Rf 0.38,hexanes-ethyl acetate 4:1); PMR (CDCl₃) 6 8.1-7.3 (m, 6, H-6 and Bz),5.8 (H-5), 5.76 (H-1′)

[0169] 2′-O-Propyluridine

[0170] A solution ofN³-(4-toluoyl)-2′-O-propyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediyluridine(27 g) in methanol (400 mL) and ammonium hydroxide (50 mL) was stirredfor 16 h at 25° C. The reaction was concentrated under reduced pressureto an oil; TLC homogenous (Rf 0.45, ethyl acetate-methanol 95:5).

[0171] The oil was dissolved in toluene (100 mL) and the solution wasevaporated under reduced pressure to dryness. The residue was dissolvedin tetrahydrofuran (300 mL). Tetrabutylammonium fluoride solution (86mL, 1 M in tetrahydrofuran) was added and the reaction was stirred at25° C. for 16 h. The pH was adjusted to 7 with Amberlite IRC-50 resin.The mixture was filtered and the resin was washed with hot methanol(2×200 mL). To the combined filtrate was added silica gel (40 g). Thesuspension was concentrated under reduced pressure to a dry powder. Theresidue was placed on top of a silica gel column (500 g) and eluted withethyl acetate and then ethyl acetate-methanol (9:1). The appropriatefractions were combined, evaporated under reduced pressure and dried at90° C./0.2 mmHg for 5 h to yield 8.0 g (70%) of light tan solid; TLCpurity 98% (Rf 0.45, ethyl acetate-methanol 4:1); PMR (DMSO) δ 11.37(H—N³), 7.9 (H-6), 5.86 (H-1′), 5.65 (H-5), 5.2 (HO-3′,5′).

[0172] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-propyluridine

[0173] 2′-O-Methyluridine (8.0 g) was evaporated under reduced pressureto an oil with pyridine (100 mL). To the residue was added4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 11.5 g, 0.34 mol) andpyridine (100 mL). The mixture was stirred at 25° C. for 1.5 h and thenquenched by the addition of methanol (10 mL) for 30 min. The mixture wasconcentrated under reduced pressure and the residue was chromatographedon silica gel (250 g, hexanes-ethyl acetate-triethylamine 80:20:1 andthen ethyl acetate-triethylamine 99:1). The appropriate fractions werecombined, evaporated under reduced pressure and dried at 25° C./0.2 mmHgfor 1 h to provide 17.4 g (100%, 30% from uridine) of tan foam; TLCpurity 98k (Rf 0.23, hexanes-ethyl acetate 4:1); PMR (DMSO) δ 11.4(H—N³), 7.78 (H-6), 7.6-6.8 (Bz), 5.8 (H-1′), 5.3 (H-5′), 5.25 (HO-3′),3.7 (CH₃O—Bz).

[0174][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-propyluridin-3′-O-yl]-N,N-diisopropylaminocyanoethoxyphosphoramidite

[0175] The product was prepared in the same manner as the adenosineanalog above by starting with intermediate5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-propyluridine and using ethylacetate-hexanes-triethylamine 59:40:1 as the chromatography eluent togive the product as a solid foam in 60% yield (18% from uridine); TLChomogenous diastereomers (Rf 0.58; 0.44, ethylacetate-hexanes-triethylamine 59:40:1); ³¹P-NMR (CDCl₃, H₃PO₄ std.) δ148.11; 148.61 (diastereomers)

[0176] C.2′-Deoxy-2′-O-methyl-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0177] Two methods will be described for the preparation of theintermediate N⁴-benzoyl-2′-O-methylcytidine. Method A involves blockingof the 3′-5′ sites with the TIPS-Cl reagent to allow alkylation only onthe 2′ position. Method B uses a direct alkylation of cytidine followedby separation of the resulting mixture. The overall yields arecomparable.

Method A

[0178] 3′,5′-O-(1,1,3,3)-Tetraisopropyl-1,3-disiloxanediylcytidine

[0179] With stirring, cytidine (40 g, 0.165 mol) and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl, 50 g, 0.159 mol)were added to dry pyridine (250 mL). After stirring for 16 h at 25° C.,the reaction was concentrated under reduced pressure to an oil. The oilwas dissolved in methylene chloride (800 mL) and washed with sat'dsodium bicarbonate (2×300 mL). The organic layer was passed through asilica gel (200 g) scrub column. The product was recovered by elutionwith methylene chloride:methanol (97:3). The appropriate fractions werecombined, evaporated under reduced pressure and dried at 25° C./0.2 mmHgfor 1 h to give 59.3 g (77%) of oil (the product may be crystallizedfrom ethyl acetate as white crystals, mp 242-244° C.); TLC purity 95%(Rf 0.59, ethyl acetate-methanol 9:1); PMR (DMSO) δ 7.7 (H-6), 5.68(H-5), 5.61 (HO-2′), 5.55 (H-1′).

[0180]N⁴-(Benzoyl)-3′-5′-O-(1,1,3,3)Tetraisopropyl-1,3-disiloxanediylcytidine

[0181] Benzoyl chloride (18.5 g, 0.13 mol) was added over 30 min to astirred solution of3′,5′-O-(1,1,3,3)-tetraisopropyl-1,3-disiloxanediylcytidine (58 g, 0.12mol) and triethylamine (15.6 g, 0.16 mol) in dimethylacetamide (400 mL)at 5° C. The mixture was allowed to warm to 25° C. for 16 h and thenpoured onto ice water (3.5 L) with stirring. The resulting solid wascollected, washed with ice water (3×500 mL) and dried at 45° C./0.2 mmHgfor 5 h to provide 77 g (100%) of solid; TLC purity ca. 90% (Rf 0.63,chloroform-methanol 9:1); PMR (CDCL₃) δ 8.32 (H-6). Lit. mp 100-101° C.

[0182]N⁴-(Benzoyl)-2′-O-methyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediylcytidine

[0183] A mixture ofN⁴-(benzoyl)-3′-5′-O-(1,1,3,3)tetra-isopropyl-1,3-disiloxanediylcytidine(166 g, 0.25 mol, 90% purity), silver oxide (150 g, 0.65 mol) andtoluene (300 mL) was evaporated under reduced pressure. More toluene(500 mL) was added and an additional amount (100 mL) was evaporated.Under a nitrogen atmosphere, methyl iodide was added in one portion andthe reaction was stirred at 40!C for 16 h. The silver salts werecollected and washed with ethyl acetate (3×150 mL). The combinedfiltrate was concentrated under reduced pressure. The residue wasdissolved in a minimum of methylene chloride, applied to a silica gelcolumn (1 kg) and eluted with hexanes-ethyl acetate (3:2®1:1). Theappropriate fractions were combined, concentrated under reduced pressureand dried at 45° C./0.2 mmHg for 1 h to yield 111 g (66%) of oil; TLCpurity ca. 90% (Rf 0.59, hexanes-ethyl acetate 3:2). PMR (CDCl₃) δ 8.8(br s, 1, H—N⁴), 8.40 (d, 1, H-6), 8.0-7.4 (m, 6, H-5 and Bz), 5.86 (s,1, H-1′), 3.74 (s, 3, CH₃O-2′).

[0184] N⁴-Benzoyl-2′-O-methylcytidine

[0185] A solution ofN⁴-(benzoyl)-2′-O-methyl-3′,5′-O-(1,1,3,3)tetraisopropyl-1,3-disiloxanediylcytidine(111 g, 0.18 mol) in methanol (160 mL) and tetrahydrofuran (640 mL) wastreated with tetrabutylammonium flouride solution (368 mL, 1 M intetrahydrofuran). The reaction was stirred at 25° C. for 16 h. The pHwas adjusted to 7 with Amberlite IRC-50 resin. The mixture was filteredand the resin was washed with hot methanol (2×200 mL). The combinedfiltrate was concentrated under reduced pressure, absorbed on silica gel(175 g) and chromatographed on silica gel (500 g, ethyl acetate-methanol19:1®4:1). Selected fractions were combined, concentrated under reducedpressure and dried at 40° C./0.2 mmHg for 2 h to yield 28 g (42.4%,21.5% from cytidine) of solid; TLC homogenous (Rf 0.37, ethyl acetate).mp 178-180° C. (recryst. from ethanol); PMR (CDCl₃) δ 11.22 (br s, 1,H—N⁴), 8.55 (d, 1, H-6), 8.1-7.2 (m, 6, H-5 and Bz), 5.89 (d, 1, H-1′),5.2 (m, 2, HO-3′,5′), 3.48 (s, 3, CH₃O-2′).

Method B

[0186] N⁴-Benzoyl-2′-O-methylcytidine

[0187] Cytidine (100 g, 0.41 mol) was dissolved in warmdimethylformamide (65° C., 1125 mL). The solution was cooled withstirring to 0° C. A slow, steady stream of nitrogen gas was deliveredthroughout the reaction. Sodium hydride (60% in oil, washed thrice withhexanes, 18 g, 0.45 mol) was added and the mixture was stirred at 0!Cfor 45 min. A solution of methyl iodide (92.25 g, 40.5 mL, 0.65 mol) indimethylformamide (400 mL) was added in portions over 4 h at 0° C. Themixture was stirred for 7 h at 25° C. and then filtered. The filtratewas concentrated to dryness under reduced pressure followed bycoevaporation with methanol (2×200 mL). The residue was dissolved inmethanol (350 mL). The solution was adsorbed on silica gel (175 g) andevaporated to dryness. The mixture was slurried in dichloromethane (500mL) and applied on top of a silica gel column (1 kg). The column waseluted with a gradient of dichloromethane-methanol (10:1®2:1). The lesspolar 2′,3′-dimethyl side product was removed and the coeluting 2′ and3′-O-methyl product containing fractions were combined and evaporatedunder reduced pressure to a syrup. The syrup was dissolved in a minimumof hot ethanol (ca. 150 mL) and allowed to cool to 25° C. The resultingprecipitate (2′ less soluble) was collected, washed with ethanol (2×25ml) and dried to give 15.2 g of pure 2′-O-methylcytidine; mp 252-254° C.(lit. mp 252-254° C.); TLC homogenous (Rf 0.50, dichloromethane-methanol3:1, (Rf of 3′ isomer is 0.50 and the dimethyl product is 0.80). Thefiltrate was evaporated to give 18 g of a mixture of isomers and sodiumiodide.

[0188] The pure 2′-O-methylcytidine (15.2 g, 0.060 mol) was dissolved ina solution of benzoic anhydride (14.7 g, 0.12 mol) in dimethylformamide(200 mL). The solution was stirred at 25° C. for 48 h and thenevaporated to dryness under reduced pressure. The residue was trituratedwith methanol (2×200 mL), collected and then triturated with warm ether(300 mL) for 10 min. The solid was collected and triturated with hot2-propanol (50 mL) and allowed to stand at 4° C. for 16 h. The solid wascollected and dried to give 17 g of product. The crude filtrate residue(18 g) of 2′-O-methylcytidine was treated with benzoic anhydride (17.3g, 0.076 mol) in dimethylformamide (250 mL) as above and triturated in asimilar fashion to give an additional 6.7 g of pure product for a totalyield of 23.7 g (16% from cytidine) of solid; TLC homogenous (Rf 0.25,chloroform-methanol 5:1, cospots with material produced from the otherroute.)

[0189]N⁴-Benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methylcytidine

[0190] N⁴-Benzoyl-2′-O-methylcytidine (28 g, 0.077 mol) was evaporatedunder reduced pressure to an oil with pyridine (400 mL). To the residuewas added 4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 28.8 g, 0.085mol) and pyridine (400 mL). The mixture was stirred at 25° C. for 2 hand then quenched by the addition of methanol (10 mL) for 30 min. Themixture was concentrated under reduced pressure and the residue waschromatographed on silica gel (500 g, hexanes-ethylacetate-triethylamine 60:40:1 and then ethyl acetate-triethylamine99:1). The appropriate fractions were combined, evaporated under reducedpressure and dried at 40° C./0.2 mmHg for 2 h to give 26 g (74%, 16%from cytidine) of foam; TLC homogenous (Rf 0.45, ethyl acetate); PMR(DMSO) δ 11.3 (H—N⁴), 8.4-6.9 (H-6, H-5, Bz), 5.95 (H-1′), 5.2 (HO-3′),3.7 (s, 6, CH₃O-trit.), 3.5 (s, 3, CH₃O-2′)

[0191] [N⁴-Benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methylcytidin-3′-O-yl]-N,N-diisopropylamino-cyanoethoxyphosphoramidite

[0192] The product was prepared in the same manner as the adenosineanalog above by starting with intermediateN-⁴-benzoyl-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-methyl-cytidineand using ethyl acetate-hexanes-triethylamine 59:40:1 as thechromatography eluent to give the product as a solid foam in 71% yield(11% from cytidine); TLC homogenous diastereomers (Rf 0.46; 0.33, ethylacetate-hexanes-triethylamine 59:40:1); ³¹P-NMR (CD₃CN, H₃PO₄ std.) δ150.34; 151.02 (diastereomers).

[0193] D.2′-Deoxy-2′-octadecyl-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0194] 2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl)purine

[0195] 2,6-Diamino-9-(β-D-ribofuranosyl)purine (50 g, 180 mmol) andsodium hydride (7 g) in DMF (1 L) were heated to boiling for 2 hr.Iodooctadecane (100 g) was added at 150° C. and the reaction mixtureallowed to cool to RT. The reaction mixture was stirred for 11 days atRT. The solvent was evaporated and the residue purified by silica gelchromatography. The product was eluted with 5% MeOH/CH₂Cl₂. Theappropriate fractions were evaporated to yield the product (11 g). ¹HNMR (DMSO-d₆) δ 0.84 (t, 3, CH₂), 1.22 (m, 32, O—CH₂—CH₂—(CH₂)₁₆), 1.86(m, 2, O—CH₂CH₂), 3.25 (m, 2, O—CH₂), 3.93 (d, 1, 4′H), 4.25 (m, 1,3′H), 4.38 (t, 1, 2′H), 5.08 (d, 1, 3′-OH), 5.48 (t, 1, 5′-OH), 5.75 (s,2, 6-NH₂), 5.84 (d, 1, 1′-H), 6.8 (s, 2, 2-NH₂), and 7.95 (s, 1, 8-H).

[0196] 2′-O-Octadecylguanosine

[0197] 2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl) purine (10 g) in0.1 M sodium phosphate buffer (50 ml, pH 7.4), 0.1 M tris buffer (1000ml, pH 7.4) and DMSO (1000 ml) was treated with adenosine deaminase (1.5g) at RT. At day 3, day 5 and day 7 an additional aliquot (500 mg, 880mg and 200 mg, respectively) of adenosine deaminase was added. Thereaction was stirred for a total of 9 day and purification by silica gelchromatography yielded the product (2 g). An analytical sample wasrecrystallized from MeOH. ¹H NMR (DMSO-d₆) δ 0.84 (t, 3, CH₃), 1.22 (s,32, O—CH₂—CH₂—(CH₂)₁₆), 5.07 (m, 2, 3′-OH and 5′-OH), 5.78 (d, 1, 1′H),6.43 (s, 2, NH₂), 7.97 (s, 1, 8H) and 10.64 (s, 1, NH₂). Anal. Calcd.for C₂₈H₄₉N₅O₅: C, 62.80; H, 9.16; N, 12.95. Found: C, 62.54; H, 9.18;N, 12.95.

[0198] N²-Isobutyryl-2′-O-octadecylguanosine

[0199] 2′-O-Octadecylguanosine (1.9 g) in pyridine (150 ml) was cooledin an ice bath, and treated with trimethylsilyl chloride (2 g, 5 eq) andisobutyryl chloride (2 g, 5 eq). The reaction mixture was stirred for 4hours, during which time it was allowed to warm to room temperature. Thesolution was cooled, water added (10 mL) and stirred for an additional30 minutes. Concentrated ammonium hydroxide (10 mL) was added and thesolution concentrated in vacuo. The residue was purified by silica gelchromatography (eluted with 3% MeOH/EtOAc) to yield 1.2 g of product.

[0200]¹H NMR (DMSO-d6) δ 0.85 (t, 3, CH₃), 1.15 (m, 38, O—CH₂CH₂(CH₂)₁₆and CH(CH₃)₂), 2.77 (m, 1, CH(CH₃)₂), 4.25 (m, 2, 2′H, 3′H), 5.08 (t, 1,5′-OH), 5.12 (d, 1, 3′-OH), 5.87 (d, 1, 1′-H), 8.27 (s, 1, 8-H), 11.68(s, 1, NH₂) and 12.08 (s, 1, NH₂). Anal. Calcd. for C₃₂H₅₅N₅O₆: C,63.47; H, 9.09; N, 11.57. Found: C, 63.53; H, 9.20; N, 11.52.

[0201] N²-Isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine

[0202] N²-Isobutyryl-2′-O-octadecylguanosine was converted to theN²-isobutyryl-5′-dimethoxytrityl-2′-O-octadecyl-guanosine as per theprocedure for adenosine above.

[0203][N²-Isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguan-3′-O-yl]-N,N-diisopropylamino-cyanoethoxyphosphoramidite

[0204] The product was prepared in the same manner as the adenosineanalog above by starting with intermediateN²-iso-butyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine.

[0205] E.N⁶-Benzoyl-2′-[N-phthalimido)prop-3-yl]5′-O(4,4′-dimethoxytrityl)]adenosine3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0206] 2′-O-[(N-Phthalimido)prop-3-yl]adenosine

[0207] The title compound was prepared as per the 2′-O-nonyladenosineprocedure using N-(3-bromopropyl)phthalimide. Chromatography on silicagel give a white solid, m.p. 123-124° C. Analysis for: C₂₁H₂₂N₆O₆.Calculated: C, 55.03; H, 4.88; N, 18.49. Found: C, 55.38; H, 4.85; N,18.46.

[0208] 2′-O-[(N-Phthalmido)prop-3-yl]-N⁶-benzoyladenosine

[0209] Benzoylation of 2′-O-[(N-phthalimido)prop-3-yl]-adenosine as perthe 2′-O-nonyladenosine procedure above give the title compound.Analysis for: C₂₈H₂₆N₆O₇. Calculated: C, 60.21; H, 4.69; N, 15.05.Found: C, 59.94; H, 4.66; N, 14.76.

[0210]2′-O-[(N-Phthalimido)prop-3-yl]-5′-O-dimethoxytrityl-N⁶-benzoyladenosine

[0211] The title compound was prepared from2′-O-[(N-phthalimido)prop-3-yl]-N⁶-benzoyladenosine as per the2′-O-nonyladenosine above. Analysis for: C₄₉H₄₄N₆O₉. Calculated: C,68.36; H, 5.15; N, 9.76. Found: C, 68.16; H, 5.03; N, 9.43.

[0212]N₆-Benzoyl-5′-O-dimethoxytrityl-2′-O-[(N-phthalimido)prop-3-yl]adenosine-3′-O,N,N-diisopropyl-β-cyanoethylphosphoramidite

[0213] The title compound was prepared from2′-O-[(N-phthalimido)prop-3-yl]-5′-O-dimethoxytrityl-N⁶-benzoyl-adenosineas above for the 2′-O-nonyladenosine compound. A white foam wasobtained.

[0214]

[0215] F.N⁶-Benzoyl-2′-[(imidazol-1-yl)butyl-4-yl]5′O-(4,4′dimethoxytrityl)]adenosine3′-O(N,N-diiso-propyl-β-cyanoethylphosphoramidite).

[0216] 2′-O-[Imidizo-1-yl-(but-4-yl)]adenosine

[0217] The title compound can be prepared as per the 2′-O-nonyladenosineprocedure using 1-(4-bromobutyl)imidazole in place of 1-bromononane.

[0218] 2′-O-[(Imidizol-1-yl)but-4-yl]-N⁶-benzoyladenosine

[0219] Benoylation of 2′-O-[(imidizol-1-yl)but-4-yl)]-adenosine as perthe 2′-O-nonyladenosine procedure above will give the title compound.

[0220]2′-O-[(Imidizol-1-yl)but-4-yl-5′-O-dimethoxytrityl-N⁶-benzoyladenosine

[0221] The title compound can be prepared from2′-O-[(imidizol-1-yl)but-4-yl]adenosine as per the 2′-O-nonyladenosineprocedure above.

[0222]N⁶-benzoyl-5′-O-dimethoxytrityl-2′-O-[(imidizol-1-yl)but-4-yl]adenosine-3′-O,N,N-diisopropyl-B-cyanoethylPhosphoramidite The title compound can be prepared from2′-O-[(imidizol-1-yl)but-4-yl)]-5′-O-dimethoxytrityl-N⁶-benzoyladenosineas per the 2′-O-nonyladenosine procedure above.

EXAMPLE 6 Preparation of 2′-Deoxy-2′-(vinyloxy) ModifiedOligonucleotides

[0223] The requisite 2′-deoxy-2′-O-vinyl ribosides of nucleic acid basesA, G, U(T), and C are prepared by modifications of literature proceduresdescribed by Sproat et al. (Nucleic Acids Research, 18, 41 (1990)] andInoue et al. [Nucleic Acids Research, 15, 6131 (1987)]. In this case1,2-dibromoethane is coupled to the 2′-hydroxyl and subsequentdehydrobromination affords the desired blocked 2′-vinyl nucleoside.Standard procedures, as described in Example 1A, are employed to preparethe 5′-DMT and 3′-phosphoramidites as listed below.

[0224] A.N⁶-Benzoyl-[2′-deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxytrityl)]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0225] B.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

[0226] C.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

[0227] D.2′-Deoxy-2′-(vinyloxy)-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

EXAMPLE 7 Preparation of 2′-Deoxy-2′-(allyloxy) ModifiedOligonucleotides

[0228] The requisite 2′-deoxy-2′-O-allyl ribosides of nucleic acid basesA, G, U(T), and C are prepared by modifications of literature proceduresdescribed by Sproat et al. [Nucleic Acids Research, 18, 41 (1990)] andInoue et al. [Nucleic Acids Research, 15, 6131 (1987)]. Standardprocedures, as described in Example 1A, are employed to prepare the5′-DMT and 3′-phosphoramidites as listed below.

[0229] A.N-Benzoyl-[2′-deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxytrityl]adenosine-3′-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

[0230] B.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-uridine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

[0231] C.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-cytidine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

[0232] D.2′-Deoxy-2′-(allyloxy)-5′-O-(4,4′-dimethoxy-trityl)-guanosine-3′-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

EXAMPLE 8 Preparation of 2′-Deoxy-2′-(methylthio), (Methylsulfinyl) and(Methylsulfonyl) Modified Oligonucleotides

[0233] A. 2′-Deoxy-2′-methylthiouridine.

[0234] 2,2′Anhydrouridine (15.5 g, 68.2 mmol) [Rao and Reese, J. Chem.Soc., Chem. Commun., 997%), methanethiol (15.7 g, 327 mmol),1,1,3,3-tetramethylguanidine (39.2 g, 341 mmol) and DMF (150 mL) wereheated at 60° C. After 12 hours, the reaction mixture was cooled andconcentrated under reduced pressure. The residual oil was purified byflash column chromatography on silica gel (300 g). Concentration of theappropriate fractions, which were eluted with CH₂Cl₂—MeOH (9:1), anddrying the residue under reduced pressure gave2′-deoxy-2′-methylthiouridine as a pale yellow solid (14.11 g, 75.4%).Attempts to crystallize the solids from EtOH-hexanes [as reported byImazawa et al., Chem. Pharm. Bull., 23, 604 (1975)] failed and thematerial turned into a hygroscopic foam.

[0235]¹H NMR (DMSO-d₆) δ 2.0 (3H, s, SCH₃), 3.34 (1H, dd, J_(3′,2′)=5.4Hz, 2′H), 3.59 (2H, br m, 5′ CH₂), 3.84 (1H, m, 4′H), 4.2 (1H, dd,J_(3′,4′)=2.2 Hz, 3′H), 5.15 (1H, t, 5′OH), 5.62 (1H, t, 3′ OH), 5.64(1H, d, J_(C6,C5)=8.2 Hz), 6.02 (1H, d, J_(1′,2′)=6 Hz, 1′H), 7.82 (1H,d, J_(C5,C6)=8.2 Hz, C6H), 11.38 (1H, br s, NH).

[0236] B. 2,2′-Anhydro-5-methyluridine.

[0237] A mixture of 5-methyluridine (16.77 g, 69.2 mmol), diphenylcarbonate (17.8 g, 83.1 mmol) and NaHCO₃ (100 mg) inhexamethylphosphoramide (175 mL) was heated to 150° C. with stirringuntil evolution of CO₂ ceased (approximately 1 hour). The reactionmixture was cooled and then poured into diethyl ether (1 L) whilestirring to furnish a brown gum. Repeated washings with diethyl ether(4×250 mL) furnished a straw-colored hygroscopic powder. The solid waspurified by short column chromatography on silica gel (400 g). Poolingand concentrating appropriate fraction, which were eluted withCH₂Cl₂—MeOH (85:15), furnished the title compound as a straw-coloredsolid (12 g, 77.3%), which crystallized from EtOH as long needles, m.p.226-227° C.

[0238] C. 2′-Deoxy-2′-methylthio-5-methyluridine.

[0239] 2,2′-Anhydro-5-methyluridine (17.02 g, 70.6 mmol), methanethiol(16.3 g, 339 mmol), 1,1,3,3-tetramethylguanidine (40.6 g, 353 mmol) andDMF (150 mL) were heated at 60° C. After 12 hours, the products werecooled and concentrated under reduced pressure. The residual oil waspurified by short silica gel column chromatography (300 g). Pooling andconcentrating appropriate fractions, which were eluted with CH₂Cl₂—MeOH(93:7), furnished the title compound as a white foam (15.08 g, 74.1%),which was crystallized from EtOH—CH₂Cl₂ as white needles.

[0240] D. 2′-Deoxy-2′-methylsulfinyluridine.

[0241] To a stirred solution of 2′-deoxy-2′-methylthiouridine (1 g, 3.65mmol) in EtOH (50 mL) was added a solution of m-chloroperbenzoic acid(50%, 1.26 g, 3.65 mmol) in EtOH (50 mL) over a period of 45 minutes at0° C. the solvent was removed under reduced pressure and the residuepurified by short silica gel (30 g) column chromatography. Concentrationof the appropriate fractions, which were eluted with CH₂Cl₂—MeOH(75:25), afforded the title compound as a white solid (0.65 g, 61.4%).Crystallization from EtOH furnished white granules, m.p. 219-221° C.

[0242]¹H NMR (DMSO-d₆) δ 2.5 (3H, s, SOCH₃), 3.56 (2H, br s, 5′CH₂), 3.8(1H, m, 4′H), 3.91 (1H, m, 2′H), 4.57 (1H, m, 3′H), 5.2 (1H, br s,5′OH), 5.75 (1H, d, C₅H), 6.19 (1H, d, 3′OH), 6.35 (1H, d, 1′H), 7.88(1H, d, C₆H), 11.43 (1H, br s, NH).

[0243] E. 2′-Deoxy-2′-methylsulfonyluridine.

[0244] To a stirred solution of 2′-deoxy-2′-methyluridine (1 g, 3.65mmol) in EtOH (50 mL) was added a solution of m-chloroperbenzoic acid(50%, 3.27 g, 14.6 mmol) in one portion at room temperature. After 2hours, the solution was filtered to separate the white precipitate whichwas formed, which upon washing (2×20 mL EtOH and 2×20 mL diethyl ether)and drying, furnished the title compound as a fine powder (0.76 g, 68%),m.p. 227-228° C.

[0245]¹H NMR (DMSO-d₆) δ 3.1 (3H, s, SO₂CH₃), 3.58 (2H, m, 5′CH₂), 3.95(1H, m, 2′H), 3.98 (1H, m, 4′H), 4.5 (1H, br s, 3′H), 5.2 (1H, br s,5′OH), 5.75 (1H, d, C₅H), 6.25 (1H, d, 3′OH), 6.5 (1H, d, 1′H), 7.8 (1H,d, C₆H), 11.45 (1H, br s, NH).

[0246] F. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine.

[0247] To a stirred solution of 2′-deoxy-2′-methylthiouridine (1.09 g, 4mmol) in dry pyridine (10 mL) was added 4,4′-dimethoxytritylchloride(1.69 g, 5 mmol) and 4-dimethylaminopyridine (50 mg) at roomtemperature. The solution was stirred for 12 hours and the reactionmixture quenched by adding MeOH (1 mL). The reaction mixture wasconcentrated under reduced pressure and the residue was dissolved inCH₂Cl₂ (100 mL), washed with saturated aqueous NaHCO₃ (2×50 mL) andsaturated aqueous NaCl (2×50 mL), and dried with MgSO₄. The solution wasconcentrated under reduced pressure and the residue purified by silicagel (30 g) column chromatography. Elution with CH₂Cl₂—MeOH-triethylamine(89:1:1) furnished the title compound as a homogenous material. Poolingand concentrating the appropriate fractions furnished the 5′-O-DMTnucleoside as a foam (1.5 g, 66.5%).

[0248]¹H NMR (DMSO-d₆) δ 2.02 (3H, s, SCH₃), 3.15-3.55 (1H, m, 2′CH),3.75 (6H, s, 2′CH₃), 3.97 (1H, m, 4′H), 4.24 (1H, m, 3′H), 5.48 (1H, d,C₅H), 5.73 (1H, d, 3′OH), 6.03 (1H, d, Cl′H), 6.82-7.4 (13H, m, ArH),6.65 (1H, d, C₆H), 11.4 (1H, br s, NH).

[0249] G.2′-Deoxy-3′-β-[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine.

[0250] To a stirred solution of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiouridine (1.5 g, 2.67mmol) in dry THF (25 mL) was added diisopropylethylamine (1.4 mL, 8mmol) and the solution was cooled to 0° C.N,N,-Diisopropyl-β-cyanoethylphosphoramidic chloride (1.26 mL, 5.34mmol) was added dropwise over a period of 15 minutes. The reactionmixture was then stirred at room temperature for 2 hours. Ethyl acetate(100 mL, containing 1% triethylamine) was added and the solution washedwith saturated NaCl (2×50 mL) and the organic layer dried over MgSO₄.The solvent was removed under reduced pressure and the residue purifiedby short silica gel (30 g) column chromatography. Elution withCH₂Cl₂—MeOH-triethylamine (98:1:1) furnished the product as a mixture ofdiastereomers. Evaporation of the appropriate fractions provided thetitle compound as a foam (1.32 g, 64.7%).

[0251]¹H NMR (CDCl₃) δ 2.0 and 2.02 (3H, s, SCH₃), 5.3 and 5.35 (1H, 2d,C₅H), 6.23 (1H, d, 1′H), 7.8 and 7.88 (1H, 2d, C₆H) and other protons.³¹P NMR (CDCl₃) δ 151.68 and 152.2 ppm.

[0252] H. 2′-Deoxy-3′,5′-di-O-acetyl-2′-methylthiouridine.

[0253] 2′-Deoxy-2′-methylthiouridine (5.0 g, 18.24 mmol) and aceticanhydride (5.6 mL, 54.74 mmol) were stirred in dry pyridine (30 mL) atroom temperature for 12 hours. The products were then concentrated underreduced pressure and the residue obtained was purified by short silicagel column chromatography. The appropriate fractions, which were elutedwith CH₂Cl₂—MeOH (9:1), were combined, evaporated under reduced pressureand the residue crystallized from EtOH to give the title compound (6.0g, 91.8%) as white needles, m.p. 132° C.

[0254]¹H NMR (CDCl₃) δ 2.17 (3H, s, SCH₃), 2.20 (6H, s, 2COCH₃), 3.40(1H, t, 2′H), 4.31-4.40 (3H, m, 4′,5′H), 5.31 (1H, m, 3′H), 5.80 (1H, d,C₅H), 6.11 (1H, d, 1′H), 7.45 (1H, d, C₆H), 8.7 (1H, br s, NH).

[0255] I.2′-Deoxy-3′,5′-di-O-acetyl-4-(1,2,4-triazol-yl)-2′-methylthiouridine.

[0256] Triethylamine (8.4 mL, 60.3 mmol) and phosphoryl chloride (1.2mL, 12.9 mmol) were added to a stirred solution of2′-deoxy-3′,5′-di-O-acetyl-2′-methylthiouridine (4.6 g, 13 mmol) in MeCN(50 mL). 1,2,4-Triazole (4.14 g, 59.9 mmol) was then added and thereactants were stirred at room temperature. After 16 hours,triethylamine-H₂O (6:1, 20 mL) was added, followed by saturated aqueousNaHCO₃ (100 mL), and the resulting mixture was extracted with CH₂Cl₂(2×100 mL). The organic layer was dried with MgSO₄ and evaporated underreduced pressure. The residue was purified by short silica gel columnchromatography. The appropriate fractions, which were eluted withCH₂Cl₂—MeOH (9:1), were evaporated under reduced pressure and theresidue was crystallized from EtOH to give the title compound (3.01 g,56.4%) as needles, m.p. 127-130° C.

[0257]¹H NMR (CDCl₃) δ 2.18 (6H, s, 2 COCH₃), 2.30 (3H, s, SCH₃), 3.67(1H, m, 2′H), 4.38-4.50 (3H, m, 4′,5′H), 5.17 (1H, t, 3′H), 6.21 (1H, d,1′H), 7.08 (1H, d, C₅H), 8.16 (1H, s, CH), 8.33 (1H, d, C₆H), 9.25 (1H,s, NH).

[0258] J. 2′-Deoxy-2′-methylthiocytidine.

[0259]2′-Deoxy-3′,5′-di-O-acetyl-4-(1,2,4-triazol-1-yl)-2′-methylthiouridine(3.0 g, 7.5 mmol) was dissolved in a saturated solution of ammonia inMeOH (70 mL) and the solution was stirred at room temperature in apressure bottle for 3 days. The products were then concentrated underreduced pressure and the residue was crystallized from EtOH—CH₂Cl₂ togive the title compound (1.06 g, 51.7%) as crystals, m.p. 201° C.

[0260]¹H NMR (DMSO-d₆) δ 1.95 (3H, s, SCH₃), 3.36 (1H, m, 2′H), 3.55(2H, m, 5′CH₂), 3.82 (1H, m, 4′H), 4.18 (1H, dd, 3′H), 5.75 (1H, d,C₅H), 6.1 (1H, d, 1′H), 7.77 (1H, d, C₆H).

[0261] Anal calcd. for C₁₀H₁₅N₃O₄S: C, 43.94; H, 5.53; N, 15.37: S,11.73. Found: C, 44.07; H, 5.45; N, 15.47; S, 11.80.

[0262] K. 2′-Deoxy-N⁴-benzoyl-2′-methylthiocytidine.

[0263] To a stirred solution of 2′-deoxy-2′-methylthiocytidine (0.86 g,3.15 mmol) in dry pyridine (20 mL) was added trimethylchlorosilane (2mL, 15.75 mmol), and stirring continued for 15 minutes. Benzoyl chloride(2.18 mL, 18.9 mmol) was added to the solution followed by stirring for2 hours. The mixture was then cooled in an ice bath and MeOH (10 mL) wasadded. After 5 minutes, ammonium hydroxide (30% aq., 20 mL) was addedand the mixture stirred for 30 minutes. The reaction mixture was thenconcentrated under reduced pressure and the residue purified by shortsilica gel (70 g) column chromatography. Elution with CH₂Cl₂—MeOH (9:1),pooling of the appropriate fractions and evaporation furnished the titlecompound (0.55 g, 46.6%), which crystallized from EtOH as needles, m.p.193-194° C.

[0264] L.N⁴-Benzoylamino-1-[2′-deoxy-5′-(4,4′-dimethoxytrityl)-2-methylthio-β-D-ribofuranosyl]pyrimidin-3(2H)-one or2′-deoxy-N⁴-benzoyl-5′-(4,4′-dimethoxytrityl)-2′-methylthiocytidine).

[0265] To a stirred solution of2′-deoxy-N⁴-benzoyl-2′-methylthiocytidine (0.80 g, 2.12 mmol) in drypyridine (10 mL) was added 4,4′-dimethoxytrityl chloride (1.16 g, 3.41mmol) and DMAP (10 mg) at room temperature. The solution was stirred for2 hours and the product concentrated under reduced pressure. The residuewas dissolved in CH₂Cl₂ (70 mL), washed with saturated NaHCO₃ (50 mL),saturated NaCl (2×50 mL), dried with MgSO₄ and evaporated under reducedpressure. The residue was purified by short silica gel (50 g) columnchromatography. Elution with CH₂Cl₂-triethylamine (99:1), pooling andconcentrating the appropriate fractions furnished the title compound(1.29 g, 90%) as a white foam.

[0266]¹H NMR (DMSO-d₆) δ 2.1 (3H, s, SCH₃), 3.5 (1H, m, 2′H), 3.75 (6H,s, OCH₃), 4.15 (1H, m, 4′H), 4.4 (1H, t, 3′H), 5.74 (1H, br d, 3′OH),6.15 (1H, d, C1H), 6.8-8.0 (25H, m, ArH and C₅H), 8.24 (1H, d, C₆H),11.3 (1H, br s, NH).

[0267] M.2′-Deoxy-N⁴-Benzoyl-3-O-[(N,N-diisopropyl)-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylthiocytidine).

[0268]2′-Deoxy-N⁴-benzoyl-5′-(4,4′-dimethoxytrityl)-2′-methylthiocytidine(1.41 g, 2.07 mmol) was treated with diisopropylethylamine (1.4 mL, 8mmol) and N,N-diisopropyl-β-cyanoethylphosphoramide chloride (1.26 mL,5.34 mmol) in dry THF (25 mL) as described in Example 8-G above. Thecrude product was purified by short silica gel (50 g) columnchromatography using CH₂Cl₂-hexanes-triethylamine (89:10:1) as theeluent. The appropriate fractions were pooled and evaporated underreduced pressure to give the title compound (1.30 g, 71%) as a whitefoam (mixture of diastereoisomers).

[0269]¹H NMR (CDCl₃) δ 2.31 (3H, s, SCH₃), 3.45-3.7 (3H, m, 2′H and5′CH₂), 3.83 (6H, m, OCH₃), 4.27-4.35 (1H, m, 4′H), 4.6-4.8 (1H, m,3′H), 6.35 (1H, 2d, 1′H), 6.82-7.8 (25H, m, ArH and C₅H), 8.38 and 8.45(1H, 2d, C₆H) and other protons. ₃₁P NMR δ 151.03 and 151.08 ppm.

[0270] N. 2′-Deoxy-2′-methylsulfinylcytidine.

[0271] 2′-Deoxy-2′-methylthiocytidine of Example 8-J was treated as perthe procedure of Example 8-D to yield the title compound as a mixture ofdiastereoisomers having a complex ¹H NMR spectrum.

[0272] O. 2′-Deoxy-2′-methylsulfonylcytidine.

[0273] 2′-Deoxy-2′-methylthiocytidine of Example 8-J was treated as perthe procedure of Example 8-E to yield the title compound.

[0274] P.N⁶-Benzoyl-3′,5′-di-O-[tetrahydropyran-2-yl]-2′-deoxy-2′-methylthioadenosine.

[0275]N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adeninefrom Example 1-D is prepared by treatment with methanethiol in thepresence of tetramethylguanidine to yield the title compound.

[0276] Q . N⁶-Benzoyl-2′-deoxy-2′-methylthioadenosine.

[0277]N⁶-Benzoyl-3′,5′-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenosinefrom Example 8-P is treated as per Example 1-F to yield the titlecompound.

[0278] R. N^(6-Benzoyl-)2′-deoxy-2′-methylsulfinyladenosine.

[0279] N⁶-Benzoyl-2′-deoxy-2′methylthioadenosine from Example 8-Q istreated as per the procedure of Example 8-D to yield the title compound.

[0280] S. N⁶-Benzoyl-2′-deoxy-2′-methylsulfonyladenosine.

[0281] N′-Benzoyl-2′-deoxy-2′methylthioadenosine from Example 8-Q istreated as per the procedure of Example 8-E to yield the title compound.

[0282] T.N²-Isobutyryl-3′,5′-di-O-(tetrahydropyran-2-yl)-2′-deoxy-2′-methylthioguanosine.

[0283]N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guaninefrom Example 1-P is treated with methanethiol in the presence of1,1,3,3-tetramethylguanidine to yield the title compound.

[0284] U. N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine.

[0285]N²-Isobutyryl-3′,5′-di-O-(tetrahydropyran-2-yl)-2′-deoxy-2′-methylthioguanosineis treated as per the procedure of Example 1-R to yield the titlecompound.

[0286] V. N²-Isobutyryl-2′-deoxy-2′-methylsulfinyl-guanosine.

[0287] N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine from Example 8-U istreated as per the procedure of Example 8-D to yield the title compound.

[0288] W. N²-Isobutyryl-2′-deoxy-2′-methylsulfonyl-guanosine.

[0289] N²-Isobutyryl-2′-deoxy-2′-methylthioguanosine from Example 8-U istreated as per the procedure of Example 8-E to yield the title compound.

[0290] X. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine.

[0291] 2′-Deoxy-2′-methylsulfinyluridine from Example 8-D above istreated as per the procedure of Example 8-F to yield the title compound.

[0292] Y.2′-Deoxy-3′-O-[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine.

[0293] 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylsulfinyluridine istreated as per the procedure of Example 8-G to yield the title compound.

[0294] Z.N⁶Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosine.

[0295] N⁶-Benzoyl-2′-deoxy-2′-methylthioadenosine from Example 8-Q aboveis treated as per the procedure of Example 8-F to yield the titlecompound.

[0296] AA. N⁶Benzoyl-2′-deoxy-3′-O-[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl-2′-methylthioadenosine.

[0297]N⁶-Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosineis treated as per the procedure of Example 8-G to yield the titlecompound.

[0298] BB.2′-Deoxy-N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioadenosine.

[0299] 2′-Deoxy-N²-isobutyryl-2′-methylthioguanosine from Example 8-Uabove is treated as per the procedure of Example 8-F to yield the titlecompound.

[0300] CC.2′-Deoxy-N²-isobutyryl-3′-O-[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide-5′-O-(4,4′-dimethoxytrityl-2′-methylthioguanosine.

[0301]2′-Deoxy-N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-methylthioguanosineis treated as per the procedure of Example 8-G to yield the titlecompound.

[0302] DD .2′-Deoxy-5′-O-(4,4′-dimethoxytrityl-2′-methylsulfonyluridine.

[0303] 2′-Deoxy-2′-methylsulfonyluridine from Example 8-E above istreated as per the procedure of Example 8-F to yield the title compound.

[0304] EE.2′-Deoxy-3′-O-[(N,N-diisopropyl)-O-β-cyanoethylphosphoramide]-5′-O-(4,4′-dimethoxytrityl-2′-methylsulfinyluridine.

[0305] 2′-Deoxy-5′-O-(4,4′dimethoxytrityl)-2′-methylsulfinyluridine istreated as per the procedure of Example 8-G to yield the title compound.

EXAMPLE 9 Chemical Conversion of an Thymine or Cytosine (Pyrimidine TypeBase) to its β-D-2′-Deoxy-2′-substituted ErythropentofuranosylNucleoside; 2′-Substituted Ribosylation)

[0306] The thymine or cytosine type analogs are trimethylsilylated understandard conditions such as hexamethyldisilazane (HMDS) and an acidcatalyst (ie. ammonium chloride) and then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythropentofuranosyl chloridein the presence of Lewis acid catalysts (i.e. stannic chloride, iodine,boron tetrafluoroborate, etc.). A specific procedure has recently beendescribed by Freskos [Nucleosides & Nucleotides, 8, 1075 (1989)] inwhich copper (I) iodide is the catalyst employed.

EXAMPLE 10 Chemical Conversion of an Adenine or Guanine (Purine TypeBase) to its β-D-2′-deoxy-2′-substituted ErythropentofuranosylNucleoside; 2′-Substituted Ribosylation)

[0307] The protected purine type analogs are converted to their sodiumsalts via sodium hydride in acetonitrile and are then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythro-pentofuranosylchloride at ambient temperature. A specific procedure has recently beendescribed by Robins et al. [Journal of American Chemical Society, 106,6379 (1984)].

EXAMPLE 11 Conversion of 2′-Deoxy-2-substituted Thymidines to theCorresponding 2′-Deoxy-2′-substituted Cytidines (Chemical Conversion ofan Pyrimidine Type 4-Keto Group to an 4-Amino Group)

[0308] The 3′ and 5′ sugar hydroxyls of the 2′modified nucleoside typesare protected by acyl groups such as toluoyl, benzoyl, p-nitrobenzoyl,acetyl, isobutryl, trifluoroacetyl, etc. under standards conditionsusing acid chlorides or anhydrides, pyridine as the solvent anddimethylaminopyridine as a catalyst. The protected nucleoside is nextchlorinated with thionyl chloride or phosphoryl chloride in pyridine oranother appropriate basic solvent. The 4-chloro group is then displacedwith ammonia in methanol. Deprotection of the sugar hydroxyls also takesplace. The amino group is benzoylated and the acyl groups areselectively removed by aqueous sodium hydroxide solution. Alternatively,the in situ process of first treating the nucleoside withchlprotrimethylsilane and base to protect the sugar hydroxyls fromsubsequent acylation may be employed. [Ogilvie, Can J. Chem., 67, 831(1989)] Another conversion approach is to replace the 4-chloro groupwith a 1,2,4-triazolo group which remains intact throughout theoligonucleotide synthesis on the automated synthesizer and is displacedby ammonia during treatment with ammonium hydroxide which cleaves theoligonucleotide from the CPG support and effects deprotection of theheterocycle. Furthermore, in many cases the 4-chloro group can beutilized as described and replaced at the end of oligonucleotidesynthesis.

EXAMPLE 12 Procedure for the Attachment of 2′-Deoxy-2′-substituted5′-Dimethoxytriphenylmethyl Ribonucleosides to the 5′-Hydroxyl ofNucleosides Bound to CPG Support

[0309] The 2′-deoxy-2′-substituted nucleoside that will reside at theterminal 3′-position of the oligonucleotide is protected as a 5′-DMTgroup (the cytosine and adenine exocyclic amino groups are benzoylatedand the guanine amino is isobutrylated) and treated with trifluoroaceticacid/bromoacetic acid mixed anhydride in pyridine anddimethylaminopyridine at 50° C. for five hours. The solution is thenevaporated under reduced pressure to a thin syrup which is dissolved inethyl acetate and passed through a column of silica gel. The homogenousfractions are collected and evaporated to dryness. A solution of 10 mLof acetonitrile, 10 μM of the 3′-O-bromomethylester-modified pyrimidinenucleoside, and 1 mL of pyridine/dimethylaminopyridine (1:1) is syringedslowly (60 to 90 sec) through a 1 μM column of CPG thymidine (AppliedBiosystems, Inc.) that had previously been treated with acid accordingto standard conditions to afford the free 5′-hydroxyl group. Othernucleoside-bound CPG columns may be employed. The eluent is collectedand syringed again through the column. This process is repeated threetimes. The CPG column is washed slowly with 10 mL of acetonitrile andthen attached to an ABI 380B nucleic acid synthesizer. Oligonucleotidesynthesis is now initiated. The standard conditions of concentratedammonium hydroxide deprotection that cleaves the thymidine ester linkagefrom the CPG support also cleaves the 3′,5′ ester linkage connecting thepyrimidine modified nucleoside to the thymidine that was initially boundto the CPG nucleoside. In this manner, any 2′-substituted nucleoside orgenerally any nucleoside with modifications in the heterocycle and/orsugar can be attached at the 3′ end of an oligonucleotide.

EXAMPLE 13 Procedure for the Conversion of 2′-Deoxy-2′-substitutedRibonucleoside-5′-DMT-3′-phosphoramidites into Oligonucleotides

[0310] The polyribonucleotide solid phase synthesis procedure of Sproatet al. [Nucleic Acids Research, 17, 3373 (1989)] is utilized to prepare2′-modified oligonucleotides.

[0311] Oligonucleotides of the sequence CGACTATGCAAGTAC (SEQ ID NO: 21)having 2′-deoxy-2′-fluoro nucleotides were incorporated at variouspositions within this sequence. In a first oligonucleotide, each of theadenosine nucleotides at positions 3, 6, 10, 11 and 14 (5′ to 3′direction) were modified to include a 2′-deoxy-2′-fluoro moiety. In afurther oligonucleotide, the adenosine and the thymidine nucleotides atpositions 3, 5, 6, 7, 10, 11, 13 and 14 were so modified. In a furtheroligonucleotide, the adenosine, thymidine and cytidine nucleotides atpositions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14 were so modified, andin even a further oligonucleotide, the nucleotides (adenosine,thymidine, cytidine and guanosine) at every position were so modified.Additionally, an oligonucleotide having the sequence CTCGTACCTTCCGGTCC(SEQ ID NO: 22) was prepared having adenosine, thymidine and cytidinenucleotides at positions 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 16also modified to contain 2′-deoxy-2′-fluoro substituents.

[0312] Various oligonucleotides were prepared incorporating nucleotideshaving 2′-deoxy-2′-methylthio substituents. For ascertaining thecoupling efficiencies of 2′-deoxy-2′-methylthio bearing nucleotides intooligonucleotides, the trimer TCC and the tetramer TUU U weresynthesized. In the trimer, the central cytidine nucleotide (the secondnucleotide) included a 2′-deoxy-2′-methylthio substituent. In thetetramer, each of the uridine nucleotides included a2′-deoxy-2′methylthio substituent. In further oligonucleotides,2′-deoxy-2′-methylthio substituent bearing nucleotides were incorporatedwithin the oligonucleotide sequence in selected sequence positions. Eachof the nucleotides at the remaining sequence positions incorporated a2′-O-methyl substituent. Thus, all the nucleotides within theoligonucleotide included a substituent group thereon, either a2′-deoxy-2′-methylthio substituent or a 2′-O-methyl substituent. Theseoligonucleotides are: GAGCUCCCAGGC (SEQ ID NO: 23) having2′-deoxy-2′-methylthio substituents at positions 4, 5, 6, 7 and 8;CGACUAUGCAAGUAC (SEQ ID NO: 24) having 2′-deoxy-2′-methylthiosubstituents at positions 1, 4, 5, 7, 9 and 13; UCCAGGUGUCCGAUC (SEQ IDNO: 25) having 2′-deoxy-2′-methylthio substituents at positions 1, 2, 3,7, 9, 10, 11 and 14; TCCAGGCCGUUUC (SEQ ID NO: 26) having2′-deoxy-2′-methylthio substituents at positions 10, 11 and 12; andTCCAGGTGTCCCC (SEQ ID NO: 27) having 2′-deoxy-2′-methylthio substituentsat positions 10, 11 and 12.

EXAMPLE 14 Preparation of 2′-Deoxy-2′-fluoro Modified PhosphorothioatesOligonucleotides

[0313] 2′-Deoxy-2′-substituted 5′-DMT nucleoside 3′-phosphoramiditesprepared as described in Examples 1-7 were inserted intosequence-specific oligonucleotide phosphorothioates as described byBeaucage et al. [Journal of American Chemical Society, 112, 1253 (1990)]and Sproat et al. [Nucleic Acids Research, 17, 3373 (1989)].

[0314] Oligonucleotides of the sequence CGA CTA TGC AAG TAC havingphosphorothioate backbone linkages and 2′-deoxy-2′-fluoro substituentbearing nucleotides were incorporated at various positions within thissequence. In a first oligonucleotide, each of the backbone linkages wasa phosphorothioate linkage and each of the adenosine, thymidine andcytidine nucleotides at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14(5′ to 3′ direction) were modified to include a 2′-deoxy-2′-fluoromoiety. In a further oligonucleotide, each of the backbone linkages wasa phosphorothioate linkage and the nucleotides (adenosine, thymidine,cytidine and guanosine) at every position were modified to include a2′-deoxy-2′-fluoro moiety.

EXAMPLE 15 Preparation of 2′-Deoxy-2′-fluoro Modified PhosphateMethylated Oligonucleotides

[0315] The protection, tosyl chloride mediated methanolysis, and milddeprotection described by Koole et al. [Journal of Organic Chemistry,54, 1657 (1989)] is applied to 2′-substituted oligonucleotides to affordphosphate-methylated 2′-substituted oligonucleotides.

EXAMPLE 16 Hybridization Analysis

[0316] A. Evaluation of the Thermodynamics of Hybridization of2′-Modified Oligonucleotides.

[0317] The ability of the 2′-modified oligonucleotides to hybridize totheir complementary RNA or DNA sequences was determined by thermalmelting analysis. The RNA complement was synthesized from T7 RNApolymerase and a template-promoter of DNA synthesized with an AppliedBiosystems, Inc. 380B RNA species was purified by ion exchange usingFPLC (LKB Pharmacia, Inc.). Natural antisense oligonucleotides or thosecontaining 2′-modifications at specific locations were added to eitherthe RNA or DNA complement at stoichiometric concentrations and theabsorbance (260 nm) hyperchromicity upon duplex to random coiltransition was monitored using a Gilford Response II spectrophotometer.These measurements were performed in a buffer of 10 mM Na-phosphate, pH7.4, 0.1 mM EDTA, and NaCl to yield an ionic strength of 10 either 0.1 Mor 1.0 M. Data was analyzed by a graphic representation of 1/T_(m) vsln[Ct], where [Ct] was the total oligonucleotide concentration. Fromthis analysis the thermodynamic parameters were determined. Based uponthe information gained concerning the stability of the duplex ofheteroduplex formed, the placement of modified pyrimidine intooligonucleotides were assessed for their effects on helix stability.Modifications that drastically alter the stability of the hybrid exhibitreductions in the free energy (delta G) and decisions concerning theirusefulness as antisense oligonucleotides were made.

[0318] As is shown in the following table (Table 1), the incorporationof 2′-deoxy-2′-fluoro nucleotides into oligonucleotides resulted insignificant increases in the duplex stability of the modifiedoligonucletide strand (the antisense strand) and its complementary RNAstrand (the sense strand). In both, phosphodiester backbone andphosphorothioate backbone oligonucleotides, the stability of the duplexincreased as the number of 2′-deoxy-2′-fluoro-containing nucleotides inthe antisense strand increased. As is evident from Table 1, withoutexception, the addition of a 2′-deoxy-2′-fluoro bearing nucleotide,irrespective of the individual substituent bearing nucleotide or theposition of that nucleotide in the oligonucleotide sequence, resulted inan increase in the duplex stability.

[0319] In Table 1, the underlined nucleotides represent nucleotides thatinclude 1 2′-deoxy-2′-fluoro substituent. The oligonucleotides prefacedwith the designation “ps” have a phosphorothioate backbone. Unlabeledoligonucleotides have phosphodiester backbones. TABLE 1 EFFECTS OF2′-DEOXY-2′-FLUORO MODIFICATIONS ON DNA (ANTISENSE) RNA (SENSE) DUPLEXSTABILITY G°37 G°37 T_(m) (° C.)/ Antisense Sequence (kcal/mol)(kcal/mol) T_(m) (° C.) T_(m) (° C.) subst. CGA CTA TGC AAG TAC −10.11 ±0.04 45.1 (SEQ ID NO:21) CGA CTA TGC AAG TAC −13.61 ± 0.08  −3.50 ± 0.0953.0 +7.9 +1.6 (SEQ ID NO:21) CGA CUA UGC AAG UAC −16.18 ± 0.08  −6.07 ±0.09 58.9 +13.8 +1.7 (SEQ ID NO:24) CGA CUA UGC AAG UAC −19.85 ± 0.05 −9.74 ± 0.06 65.2 +20.1 +1.8 (SEQ ID NO:24) ps(CGA CTA TGC AAG TAC) −7.58 ± 0.06 33.9 −11.2 (SEQ ID NO:21) ps(CGA CUA UGC AAG UAC) −15.90 ±0.34  −8.32 ± 0.34 60.9 +27.0 +2.5 (SEQ ID NO:24) CTC GTA CCT TCC GGT CC−14.57 ± 0.13 61.6 (SEQ ID NO:22) CUC GUA CCU UCC GGU CC −27.81 ± 0.05−13.24 ± 0.14 81.6 +1.4 (SEQ ID NO:28)

[0320] As is evident from Table 1, the duplexes formed between RNA andoligonucleotides containing 2′-deoxy-2′-fluoro substituted nucleotidesexhibited increased binding stability as measured by the hybridizationthermodynamic stability. Delta T_(m)s of greater than 20° C. weremeasured. By modifying the backbone to a phosphorothioate backbone, evengreater delta T_(m)s were observed. In this instance, delta T_(m)sgreater than 31° C. were measured. These fluoro-substitutedoligonucleotides exhibited a consistent and additive increase in thethermodynamic stability of the duplexes formed with RNA. While we do notwish to be bound by theory, it is presently believed that the presenceof a 2′-fluoro substituent results in the sugar moiety of the2′-fluoro-substituted nucleotide assuming substantially a 3′-endoconformation and this results in the oligonucleotide-RNA complexassuming an A-type helical conformation.

[0321] B. Fidelity of Hybridization of 2′-Modified Oligonucleotides

[0322] The ability of the 2′-modified antisense oligonucleotides tohybridize with absolute specificity to the targeted mRNA was shown byNorthern blot analysis of purified target mRNA in the presence of totalcellular RNA. Target mRNA was synthesized from a vector containing thecDNA for the target mRNA located downstream from a T7 RNA polymerasepromoter. Synthesized mRNA was electrophoresed in an agarose gel andtransferred to a suitable support membrane (i.e. nitrocellulose). Thesupport membrane was blocked and probed using ³²P-labeled antisenseoligonucleotides. The stringency will be determined by replicate blotsand washing in either elevated temperatures or decreased ionic strengthof the wash buffer. Autoradiography was performed to assess the presenceof heteroduplex formation and the autoradiogram quantitated by laserdensitometry (LKB Pharmacia, Inc.). The specificity of hybrid formationwas determined by isolation of total cellular RNA by standard techniquesand its analysis by agarose electrophoresis, membrane transfer andprobing with the labeled 2′-modified oligonucleotides. Stringency waspredetermined for the unmodified antisense oligonucleotides and theconditions used such that only the specifically targeted mRNA wascapable of forming a heteroduplex with the 2′-modified oligonucleotide.

[0323] C. Base-pair Specificity of Oligonucleotides and RNA

[0324] Base-pair specificity of 2′-deoxy-2′-fluoro modifiedoligonucleotides with the RNA complement (a “Y” strand) was determinedby effecting single base-pair mismatches and a bulge. The results ofthese determinations are shown in Table 2. An 18mer “X” strandoligonucleotide containing 14 adenosine, thymidine and cytidinenucleotides having a 2′-deoxy-2′-fluoro substituent was hybridized withthe RNA complement “Y” strand in which the 10th position was varied. InTable 2, the underlined nucleotides represent nucleotides that include a2′-deoxy-2′-fluoro substituent.

[0325] As is evident from Table 2, the 2′-deoxy-2′-fluoro modifiedoligonucleotide formed a duplex with the RNA complement with greaterspecificity than a like-sequenced unmodified oligonucleotide. TABLE 2EFFECTS OF SINGLE BASE MISMATCHES ON 2′-DEOXY-2′-FLUORO MODIFIED DNA-RNADUPLEX STABILITY G°37 G°37 T_(m) T_(m) Y Base pair type (kcal/mol)(kcal/mol) (° C.) (° C.) X strand: deoxy(CTC GTA CCT TTC CGG TCC) (SEQID NO: 29) Y strand: ribo(^(3′)GAG CAU GGY AAG GCC AGG^(5′)) (SEQ ID NO:30) A Watson-Crick −14.57 ± 0.13 61.6 C T-C mismatch −12.78 ± 0.11   1.79 ± 0.17 54.4 −7.2 G T-G mismatch −16.39 ± 0.25  −1.82 ± 0.28 61.70.1 U T-U mismatch −13.48 ± 0.17    1.09 ± 0.22 55.9 −5.7 None Bulged T−14.86 ± 0.35 −0.284 ± 0.37 59.4 −2.2 X strand: deoxy(CUC GUA CCU UUCCGG UCC) (SEQ ID NO: 31) Y strand: ribo(^(3′)GAG CAU GGY AAG GCCAGG^(5′)) (SEQ ID NO: 30) A Watson-Crick −27.80 ± 0.05 81.6 C U-Cmismatch −21.98 ± 0.28    5.82 ± 0.28 73.8 −7.8 G U-G mismatch −21.69 ±0.16    6.12 ± 0.17 77.8 −3.8 U U-U mismatch −18.68 ± 0.15    9.13 ±0.16 73.6 −8.0 None Bulged U −22.87 ± 0.27    4.94 ± 0.27 75.5 −6.2

EXAMPLE 17 Nuclease Resistance

[0326] A. Evaluation of the Resistance of 2′-Modified Oligonucleotidesto Serum and Cytoplasmic Nucleases.

[0327] Natural phosphorothioate, and 2-modified oligonucleotides wereassessed for their resistance to serum nucleases by incubation of theoligonucleotides in media containing various concentrations of fetalcalf serum or adult human serum. Labeled oligonucleotides were incubatedfor various times, treated with protease K and then analyzed by gelelectrophoresis on 20% polyacrylamide-urea denaturing gels andsubsequent autoradiography. Autoradiograms were quantitated by laserdensitometry. Based upon the location of the modifications and the knownlength of the oligonucleotide it was possible to determine the effect onnuclease degradation by the particular 2′-modification. For thecytoplasmic nucleases, a HL60 cell line was used. A post-mitochondrialsupernatant was prepared by differential centrifugation and the labeledoligonucleotides were incubated in this supernatant for various times.Following the incubation, oligonucleotides were assessed for degradationas outlined above for serum nucleolytic degradation. Autoradiographyresults were quantitated for comparison of the unmodified, thephosphorothioates, and the 2′-modified oligonucleotides.

[0328] Utilizing these test systems, the stability of a 15 meroligonucleotide having 2′-deoxy-2′-fluoro-substituted nucleotides atpositions 12 and 14 and a phosphorothioate backbone were investigated.As a control, an unsubstituted phosphodiester oligonucleotide was 50%degraded within 1 hour, and 100% degraded within 20 hours. Incomparison, for the 2′-deoxy-2′-fluoro-substituted oligonucleotidehaving a phosphorothioate backbone, degradation was limited to less that10% after 20 hours.

[0329] B. Evaluation of the Resistance of 2′-Modified Oligonucleotidesto Specific Endo- and Exonucleases.

[0330] Evaluation of the resistance of natural and 2′-modifiedoligonucleotides to specific nucleases (i.e., endonucleases, 3′,5′-exo-,and 5′,3′-exonucleases) was done to determine the exact effect of themodifications on degradation. Modified oligonucleotides were incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea was added andanalysis on 20% poly-acrylamide gels containing urea was done. Gelproducts were visualized by staining using Stains All (Sigma ChemicalCo.). Laser densitometry was used to quantitate the extend ofdegradation. The effects of the 2′-modifications were determined forspecific nucleases and compared with the results obtained from the serumand cytoplasmic systems.

EXAMPLE 18 Oligonucleotide Synthesis

[0331] Unsubstituted and substituted oligonucleotides were synthesizedon an automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine. Forphosphorothioate oligonucleotides, the standard oxidation bottle wasreplaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxide inacetonitrile for the step wise thiation of the phosphite linkages. Thethiation wait step was increased to 68 sec and was followed by thecapping step. After cleavage from the CPG column and deblocking inconcentrated ammonium hydroxide at 55° C. (18 hours), theoligonucleotides were purified by precipitating twice with 2.5 volumesof ethanol from a 0.5 M NaCl solution. Analytical gel electrophoresiswas accomplished in 20% acrylamide, 8 M urea, 454 mM Tris-borate buffer,pH=7.0. Oligonucleotides and phosphorothioates were judged, based onpolyacrylamide gel electrophoresis, to be greater than 80% full-lengthmaterial.

EXAMPLE 19 Oligonucleotide Having 2′-Substituted OligonucleotidesRegions Flanking Central 2′-Deoxy Phosphorothioate OligonucleotideRegion

[0332] A 15 mer RNA target of the sequence 5′GCGTTTTTTTTTTGCG 3′ (SEQ IDNO: 32) was prepared in the normal manner on the DNA sequencer using RNAprotocols. A series of complementary phosphorothioate oligonucleotideshaving 2′-O-substituted nucleotides in regions that flank a 2′-deoxyregion were prepared utilizing 2′-O-substituted nucleotide precursorsprepared as per known literature preparations, i.e. 2′-O-methyl, or asper the procedure of International Publication Number WO 92/03568,published Mar. 5, 1992. The 2′-O-substituted nucleotides were added astheir 5′-O-dimethoxytrityl-3′-phosphoramidites in the normal manner onthe DNA synthesizer. The complementary oligonucleotides have thesequence of 5′ CGCAAAAAAAAAAAAACGC 3′ (SEQ ID NO: 33). The2′-O-substituent was located in CGC and CG regions of theseoligonucleotides. The following 2′-O-substituents were used: 2′-fluoro;2′-O-methyl; 2′-O-propyl; 2′-O-allyl; 2′-O-aminopropoxy;2′-O-(methoxyethoxyethyl), 2′-O-imidazolebutoxy and2′-O-imidazolepropoxy.

EXAMPLE 20 Ras-Luciferase Reporter Gene Assembly

[0333] The ras-luciferase reporter genes described in this study wereassembled using PCR technology. Oligonucleotide primers were synthesizedfor use as primers for PCR cloning of the 5′-regions of exon 1 of boththe mutant (codon 12) and non-mutant (wild-type) human H-ras genes.H-ras gene templates were purchased from the American Type CultureCollection (ATCC numbers 41000 and 41001) in Bethesda, Md. Theoligonucleotide PCR primers5′-ACA-TTA-TGC-TAG-CTT-TTT-GAG-TAA-ACT-TGT-GGG-GCA-GGA-GAC-CCT-GT-3′(sense) (SEQ ID NO: 34), and5′-GAG-ATC-TGA-AGC-TTC-TGG-ATG-GTC-AGC-GC-3′ (antisense) (SEQ ID NO:35), were used in standard PCR reactions using mutant and non-mutantH-ras genes as templates. These primers are expected to produce a DNAproduct of 145 base pairs corresponding to sequences −53 to +65(relative to the translational initiation site) of normal and mutantH-ras, flanked by NheI and HindIII restriction endonuclease sites. ThePCR product was gel purified, precipitated, washed and resuspended inwater using standard procedures.

[0334] PCR primers for the cloning of the P. pyralis (firefly)luciferase gene were designed such that the PCR product would code forthe full-length luciferase protein with the exception of theamino-terminal methionine residue, which would be replaced with twoamino acids, an amino-terminal lysine residue followed by a leucineresidue. The oligonucleotide PCR primers used for the cloning of theluciferase gene were5′-GAG-ATC-TGA-AGC-TTG-AAG-ACG-CCA-AAA-ACA-TAA-AG-3′ (sense) (SEQ ID NO:36), and 5′-ACG-CAT-CTG-GCG-CGC-CGA-TAC-CGT-CGA-CCT-CGA-3′ (antisense)(SEQ ID NO: 37), were used in standard PCR reactions using acommercially available plasmid (pT3/T7-Luc) (Clontech), containing theluciferase reporter gene, as a template. These primers were expected toyield a product of approximately 1.9 kb corresponding to the luciferasegene, flanked by HindIII and BssHII restriction endonuclease sites. Thisfragment was gel purified, precipitated, washed and resuspended in waterusing standard procedures.

[0335] To complete the assembly of the ras-luciferase fusion reportergene, the ras and luciferase PCR products were digested with theappropriate restriction endonucleases and cloned by three-part ligationinto an expression vector containing the steroid-inducible mouse mammarytumor virus promotor MMTV using the restriction endonucleases NheI,HindIII and BssHII. The resulting clone results in the insertion ofH-ras 5′ sequences (−53 to +65) fused in frame with the fireflyluciferase gene. The resulting expression vector encodes aras-luciferase fusion product which is expressed under control of thesteroid-inducible MMTV promoter.

EXAMPLE 21 Transfection of Cells With Plasmid DNA

[0336] Transfections were performed as described by Greenberg in CurrentProtocols in Molecular Biology, Ausubel et al., Eds., John Wiley andSons, New York, with the following modifications: HeLa cells were platedon 60 mm dishes at 5×10⁵ cells/dish. A total of 10 μg of DNA was addedto each dish, of which 9 μg was ras-luciferase reporter plasmid and 1 μgwas a vector expressing the rat glucocorticoid receptor under control ofthe constitutive Rous sarcoma virus (RSV) promoter. Calciumphosphate-DNA coprecipitates were removed after 16-20 hours by washingwith Tris-buffered saline [50 Mm Tris-Cl (pH 7.5), 150 mM NaCl]containing 3 mM EGTA. Fresh medium supplemented with 10% fetal bovineserum was then added to the cells. At this time, cells were pretreatedwith antisense oligonucleotides prior to activation of reporter geneexpression by dexamethasone.

EXAMPLE 22 Oligonucleotide Treatment of Cells

[0337] Immediately following plasmid transfection, cells were thricewashed with OptiMEM (GIBCO), and prewarmed to 37° C. 2 ml of OptiMEMcontaining 10 μg/mlN-[1-(2,3-diolethyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA)(Bethesda Research Labs, Gaithersburg, Md.) was added to each dish andoligonucleotides were added directly and incubated for 4 hours at 37° C.OptiMEM was then removed and replaced with the appropriate cell growthmedium containing oligonucleotide. At this time, reporter geneexpression was activated by treatment of cells with dexamethasone to afinal concentration of 0.2 μM. Cells were harvested 12-16 hoursfollowing steroid treatment.

EXAMPLE 23 Luciferase Assays

[0338] Luciferase was extracted from cells by lysis with the detergentTriton X-100, as described by Greenberg in Current Protocols inMolecular Biology, Ausubel et al., Eds., John Wiley and Sons, New York.A Dynatech ML1000 luminometer was used to measure peak luminescence uponaddition of luciferin (Sigma) to 625 μM. For each extract, luciferaseassays were performed multiple times, using differing amounts of extractto ensure that the data were gathered in the linear range of the assay.

EXAMPLE 24 Antisense Oligonucleotide Inhibition of ras-Luciferase GeneExpression

[0339] A series of antisense phosphorothioate oligonucleotide analogstargeted to the codon-12 point mutation of activated H-ras were testedusing the ras-luciferase reporter gene system described in the foregoingexamples. This series comprised a basic sequence and analogs of thatbasic sequence. The basic sequence was of known activity as reported inInternational Publication Number WO 92/22651 identified above. In boththe basic sequence and its analogs, each of the nucleotide subunitsincorporated phosphorothioate linkages to provide nuclease resistance.Each of the analogs incorporated nucleotide subunits that contained2′-O-methyl substitutions and 2′-deoxy-erythro-pentofuranosyl sugars. Inthe analogs, a subsequence of the 2′-deoxy-erythro-pentofuranosylsugar-containing subunits was flanked on both ends by subsequences of2′-O-methyl substituted subunits. The analogs differed from one anotherwith respect to the length of the subsequence of the2′-deoxy-erythro-pentofuranosyl sugar containing nucleotides. The lengthof these subsequences varied by 2 nucleotides between 1 and 9 totalnucleotides. The 2′-deoxy-erythro-pentofuranosyl nucleotidesub-sequences were centered at the point mutation of the codon-12 pointmutation of the activated ras.

[0340] The base sequences, sequence reference numbers and sequence IDnumbers of these oligonucleotides (all are phosphorothioate analogs) areshown in Table 3. In this table those nucleotides identified with a“^(M)” contain a 2′-O-methyl substituent group and the remainder of thenucleotides identified with a “_(d)” are 2′-deoxy-erythro-pentofuranosylnucleotides. TABLE 3 Chimeric 2′-O-methyl P = S oligonucleotides SEQ IDOLIGO SEQUENCE NO: 2570 C_(d)C_(d)A_(d) C_(d)A_(d)C_(d) C_(d)G_(d)A_(d)C_(d)G_(d)G_(d) C_(d)G_(d)C_(d) C_(d)C_(d) 1 3975 C^(M)C^(M)A^(M)C^(M)A^(M)C^(M) C^(M)G^(M)A_(d) C^(M)G^(M)G^(M) C^(M)G^(M)C^(M) 1C^(M)C^(M) 3979 C^(M)C^(M)A^(M) C^(M)A^(M)C^(M) C^(M)G_(d)A_(d)C_(d)G^(M)G^(M) C^(M)G^(M)C^(M) C^(M)C^(M) 1 3980 C^(M)C^(M)A^(M)C^(M)A^(M)C^(M) C_(d)G_(d)A_(d) C_(d)G_(d)G^(M) C^(M)G^(M)C^(M)C^(M)C^(M) 1 3985 C^(M)C^(M)A^(M) C^(M)A^(M)C_(d) C_(d)G_(d)A_(d)C_(d)G_(d)G_(d) C^(M)G^(M)C^(M) C^(M)C^(M) 1 3984 C^(M)C^(M)A^(M)C^(M)A_(d)C_(d) C_(d)G_(d)A_(d) C_(d)G_(d)G_(d) C_(d)G^(M)C^(M)C^(M)C^(M) 1

[0341]FIG. 1 shows dose-response data in which cells were treated withthe phosphorothioate oligonucleotides of Table 3. Oligonucleotide 2570is targeted to the codon-12 point mutation of mutant (activated) H-rasRNA. The other nucleotides have 2′-O-methyl substituents groups thereonto increase binding affinity with sections of various lengths ofinterspaced 2′-deoxy-erythro-pentofuranosyl nucleotides. The controloligonucleotide is a random phosphorothioate oligonucleotide analog, 20bases long. Results are expressed as percentage of luciferase activityin transfected cells not treated with oligonucleotide. As the figureshows, treatment of cells with increasing concentrations ofoligonucleotide 2570 resulted in a dose-dependent inhibition ofras-luciferase activity in cells expressing the mutant form ofras-luciferase. Oligonucleotide 2570 displays an approximate threefoldselectivity toward the mutant form of ras-luciferase as compared to thenormal form. As is further seen in FIG. 1, each of the oligonucleotides3980, 3985 and 3984 exhibited greater inhibition of ras-luciferaseactivity than did oligonucleotide 2570. The greatest inhibition wasdisplayed by oligonucleotide 3985 that has a subsequence of2′-deoxy-erythro-pentofuranosyl nucleotides seven nucleotides long.oligonucleotide 3980, having a five nucleotide long2′-deoxy-erythro-pentofuranosyl nucleotide subsequence exhibited thenext greatest inhibition followed by oligonucleotide 3984 that has anine nucleotide 2′-deoxy-erythro-pentofuranosyl nucleotide subsequence.

[0342]FIG. 2 shows the results similar to FIG. 1 except it is in bargraph form. Further seen on FIG. 2 is the activity of oligonucleotide3975 and oligonucleotide 3979. These oligonucleotides have subsequencesof 2′-deoxy-erythro-pentofuranosyl nucleotides one and three nucleotideslong, respectively. As is evident from FIG. 2, neither of theoligonucleotides having either the one or the three2′-deoxy-erythro-pentofuranosyl nucleotide subsequences showedsignificant activity. There was measurable activity for the threenucleotide subsequence oligonucleotide 3979 at the highest concentrationdose.

[0343] The increases in activity of oligonucleotides 3980, 3985 and 3984compared to oligonucleotide 2570 is attributed to the increase inbinding affinity imparted to these compounds by the 2′-O-methylsubstituent groups located on the compounds and by the RNase Hactivation imparted to these compounds by incorporation of a subsequenceof 2′-deoxy-erythro-pentofuranosyl nucleotides within the main sequenceof nucleotides. In contrast to the active compounds of the invention, itis interesting to note that sequences identical to those of the activeoligonucleotides 2570, 3980, 3985 and 3984 but having phosphodiesterlinkages in stead of the phosphorothioate linkages of the activeoligonucleotides of the invention showed no activity. This is attributedto these phosphodiester compounds being substrates for nucleases thatdegrade such phosphodiester compounds thus preventing them potentiallyactivating RNase H. Other sugar modifications: The effects of other 2′sugar modifications besides 2′-O-methyl on antisense activity inchimeric oligonucleotides have been examined. These modifications arelisted in Table 4, along with the T_(m) values obtained when 17 meroligonucleotides having 2′-modified nucleotides flanking a 7-base deoxygap were hybridized with a 25 mer oligoribonucleotide complement asdescribed in Example 25. A relationship was observed for theseoligonucleotides between alkyl length at the 2′ position and T_(m). Asalkyl length increased, T_(m) decreased. The 2′-fluoro chimericoligonucleotide displayed the highest T_(m) of the series. TABLE 4Correlation of T_(m) with Antisense Activity 2′-modified 17-mer with7-deoxy gap CCACACCGACGGCGCCC (SEQ ID NO: 1) 2′ MODIFICATIONT_(m (° C.)) IC₅₀ (nM) Deoxy 6 4.2 150 O-Pentyl 6 8.5 150 O-Propyl 7 0.470 O-Methyl 7 4.7 20 Fluoro 7 6.9 10

[0344] These 2′ modified oligonucleotides were tested for antisenseactivity against H-ras using the transactivation reporter gene assaydescribed in Example 26. All of these 2′ modified chimeric compoundsinhibited ras expression, with the 2′-fluoro 7-deoxy-gap compound beingthe most active. A 2′-fluoro chimeric oligonucleotide with a centered5-deoxy gap was also active.

[0345] Chimeric phosphorothioate oligonucleotides having SEQ ID NO: 1having 2′-O-propyl regions surrounding a 5-base or 7-base deoxy gap werecompared to 2′-O-methyl chimeric oligonucleotides. ras expression in T24cells was inhibited by both 2′-O-methyl and 2′-O-propyl chimericoligonucleotides with a 7-deoxy gap and a uniform phosphorothioatebackbone. When the deoxy gap was decreased to five nucleotides, only the2′-O-methyl oligonucleotide inhibited ras expression.

[0346] Antisense oligonucleotide inhibition of H-ras gene expression incancer cells: Two phosphorothioate oligonucleotides (2502, 2503)complementary to the ras AUG region were tested as described in Example27, along with chimeric oligonucleotides (4998, 5122) having the samesequence and 7-base deoxy gaps flanked by 2′-O-methyl regions. Thesechimeric oligonucleotides are shown in Table 5. TABLE 5 Chimericphosphorothioate oligonucleotides having 2′-O-methyl ends (bold) andcentral deoxy gap (AUG target) OLIGO # DEOXY SEQUENCE SEQ ID NO: 2502 20C T T A T A T T C C G T C A T C G C T C 2 4998 7 C T T A T AT T C C G TC A T C G C T C 2 2503 20 T C C G T C AT C G C T C C T C A G G G 3 51227 T C C G T C AT C G C T C C T C A G G G 3

[0347] Compound 2503 inhibited ras expression in T24 cells by 71%, andthe chimeric compound (4998) inhibited ras mRNA even further (84%inhibition). Compound 2502, also complementary to the AUG region,decreased ras RNA levels by 26% and the chimeric version of thisoligonucleotide (5122) demonstrated 15% inhibition. Also included inthis assay were two oligonucleotides targeted to the mutant codon 12.Compound 2570 (SEQ ID NO: 1) decreased ras RNA by 82% and the2′-O-methyl chimeric version of this oligonucleotide with a seven-deoxygap (3985) decreased ras RNA by 95%.

[0348] Oligonucleotides 2570 and 2503 were also tested to determinetheir effects on ras expression in HeLa cells, which have a wild-type(i.e., not activated) H-ras codon-12. While both of theseoligonucleotides inhibited ras expression in T24 cells (having activatedcodon-12), only the oligonucleotide (2503) specifically hybridizablewith the ras AUG inhibited ras expression in HeLa cells. Oligonucleotide2570 (SEQ ID NO: 1), specifically hybridizable with the activatedcodon-12, did not inhibit ras expression in HeLa cells, because thesecells lack the activated codon-12 target.

[0349] Oligonucleotide 2570, a 17 mer phosphorothioate oligonucleotidecomplementary to the codon-12 region of activated H-ras, was tested forinhibition of ras expression (as described in Example 25) in T24 cellsalong with chimeric phosphorothioate 2′-O-methyl oligonucleotides 3980,3985 and 3984, which have the same sequence as 2570 and have deoxy gapsof 5, 7 and 9 bases, respectively (shown in Table 3). The fully 2′-deoxyoligonucleotide 2570 and the three chimeric oligonucleotides decreasedras mRNA levels in T24 cells. Compounds 3985 (7-deoxy gap) and 3984(9-deoxy gap) decreased ras mRNA by 81%; compound 3980 (5-deoxy gap)decreased ras mRNA by 61%. Chimeric oligonucleotides having thissequence, but having 2′-fluoro-modified nucleotides flanking a 5-deoxy(4689) or 7-deoxy (4690) gap, inhibited ras mRNA expression in T24cells, with the 7-deoxy gap being preferred (82% inhibition, vs 63%inhibition for the 2′-fluoro chimera with a 5-deoxy gap).

[0350] Antisense oligonucleotide inhibition of proliferation of cancercells: Three 17mer oligonucleotides having the same sequence (SEQ ID NO:1), complementary to the codon 12 region of activated ras, were testedfor effects on T24 cancer cell proliferation as described in Example 28.3985 is a full phosphorothioate oligonucleotide having a 7-deoxy gapflanked by 2′-O-methyl nucleotides, and 4690 is a full phosphorothioateoligonucleotide having a 7-deoxy gap flanked by 2′-F nucleotides(C^(F)C^(F)A^(F) C^(F)A^(F)C_(d) C_(d)G_(d)A_(d) C_(d)G_(d)G_(d)C^(F)G^(F)C^(F) C^(F)C^(F), SEQ ID NO: 1, nucleotides identified with an“^(F)” contain a 2′-O-fluoro substituent group and the remainder of thenucleotides identified with a “_(d)” are 2′-deoxy-erythro-pentofuranosylnucleotides). Effects of these oligonucleotides on cancer cellproliferation correlated well with their effects on ras mRNA expressionshown by Northern blot analysis: oligonucleotide 2570 inhibited cellproliferation by 61%, the 2′-O-methyl chimeric oligonucleotide 3985inhibited cell proliferation by 82%, and the 2′-fluoro chimeric analoginhibited cell proliferation by 93%.

[0351] In dose-response studies of these oligonucleotides on cellproliferation, the inhibition was shown to be dose-dependent in the 25nM-100 nM range. IC₅₀ values of 44 nM, 61 nM and 98 nM could be assignedto oligonucleotides 4690, 3985 and 2570, respectively. The randomoligonucleotide control had no effect at the doses tested.

[0352] The effect of ISIS 2570 on cell proliferation was celltype-specific. The inhibition of T24 cell proliferation by thisoligonucleotide was four times as severe as the inhibition of HeLa cellsby the same oligonucleotide (100 nM oligonucleotide concentration). ISIS2570 is targeted to the activated (mutant) ras codon-12, which ispresent in T24 but lacking in HeLa cells, which have the wild-typecodon-12.

[0353] Chimeric backbone-modified oligonucleotides: Oligonucleotidesdiscussed in previous examples have had uniform phosphorothioatebackbones. The 2′modified chimeric oligonucleotides discussed above arenot active in uniform phosphodiester backbones. A chimericoligonucleotide was synthesized (ISIS 4226) having 2′-O-methyl regionsflanking a 5-nucleotide deoxy gap, with the gap region having a P═Sbackbone and the flanking regions having a P═O backbone. Anotherchimeric oligonucleotide (ISIS 4223) having a P═O backbone in the gapand P═S in flanking regions was also made. These oligonucleotides areshown in Table 6.

[0354] Additional oligonucleotides were synthesized, completely 2′deoxyand having phosphorothioate backbones containing either a singlephosphodiester (ISIS 4248), two phosphodiesters (ISIS 4546), threephosphodiesters (ISIS 4551), four phosphodiesters (ISIS 4593), fivephosphodiesters (ISIS 4606) or ten phosphodiester linkages (ISIS-4241)in the center of the molecule. These oligonucleotides are also shown inTable 6. TABLE 6 Chimeric backbone (P = S/P = O) oligonucleotides having2′-O-methyl wings (bold) and central deoxy gap (backbone linkagesindicated by s (P = S) or o (P = O) SEQ # ID OLIGO P = S SEQUENCE NO:2570 16 CsCsAsCsAsCsCsGsAsCsGsGsCsGsCsCsC 1 4226 5CoCoAoCoAoCsCsGsAsCsGoGoCoGoCoCoC 1 4233 11CsCsAsCsAsCoCoGoAoCoGsGsCsGsCsCsC 1 4248 15CsCsAsCsAsCsCsGsAoCsGsGsCsGsCsCsC 1 4546 14CsCsAsCsAsCsCsGoAoCsGsGsCsGsCsCsC 1 4551 13CsCsAsCsAsCsCsGoAoCoGsGsCsGsCsCsC 1 4593 12CsCsAsCsAsCsCoGoAoCoGsGsCsGsCsCsC 1 4606 11CsCsAsCsAsCsCoGoAoCoGoGsCsGsCsCsC 1 4241 6CsCsAsCoAoCoCoGoAoCoGoGoCoGsCsCsC 1

[0355] Oligonucleotides were incubated in crude HeLa cellular extractsat 37° C. to determine their sensitivity to nuclease degradation asdescribed in Dignam et al. [Nucleic Acids Res., 11, 1475 (1983)]. Theoligonucleotide (4233) with a 5-diester gap betweenphosphorothioate/2′-O-methyl wings had a T½ of 7 hr. The oligonucleotidewith a five-phosphorothioate gap in a phosphorothioate/2′-O-methylmolecule had a T½ of 30 hours. In the set of oligonucleotides having oneto ten diester linkages, the oligonucleotide (4248) with a singlephosphodiester linkage was as stable to nucleases as was thefull-phosphorothioate molecule, ISIS 2570, showing no degradation after5 hours in HeLa cell extract. Oligonucleotides with two-, three andfour-diester gaps had T½ of approximately 5.5 hours, 3.75 hours, and 3.2hours, and oligonucleotides with five or ten deoxy linkages had T½ of1.75 hours and 0.9 hours, respectively.

[0356] Antisense activity of chimeric backbone-modifiedoligonucleotides: A uniform phosphorothioate backbone is not requiredfor antisense activity. ISIS 4226 and ISIS 4233 were tested in theras-luciferase reporter system for effect on ras expression along withISIS 2570 (fully phosphorothioate/all deoxy), ISIS 3980 (fullyphosphorothioate, 2′-O-methyl wings with deoxy gap) and ISIS 3961 (fullyphosphodiester, 2′-O-methyl wings with deoxy gap). All of theoligonucleotides having a P═S (i.e., nuclease-resistant) gap regioninhibited ras expression. The two completely 2′deoxy oligonucleotideshaving phosphorothioate backbones containing either a singlephosphodiester (ISIS 4248) or ten phosphodiester linkages (ISIS 4241) inthe center of the molecule were also assayed for activity. The compoundcontaining a single P═O was just as active as a full P═S molecule, whilethe same compound containing ten P═O was completely inactive.

[0357] Chimeric phosphorothioate oligonucleotides of SEQ ID NO: 1 weremade, having a phosphorothioate backbone in the 7-base deoxy gap regiononly, and phosphodiester in the flanking regions, which were either2′-O-methyl or 2′-O-propyl. The oligonucleotide with the 2′-O-propyldiester flanking regions was able to inhibit ras expression.

EXAMPLE 25 Melting Curves

[0358] Absorbance vs. temperature curves were measured at 260 nm using aGilford 260 spectrophotometer interfaced to an IBM PC computer and aGilford Response II spectrophotometer. The buffer contained 100 mM Na⁺,10 mM phosphate and 0.1 mM EDTA, pH 7. Oligonucleotide concentration was4 μM each strand determined from the absorbance at 85° C. and extinctioncoefficients calculated according to Puglisi and Tinoco [Methods inEnzymol., 180, 304 (1989). T_(m) values, free energies of duplexformation and association constants were obtained from fits of data to atwo state model with linear sloping baselines. [Petersheim and Turner,Biochemistry, 22, 256 (1983). Reported parameters are averages of atleast three experiments. For some oligonucleotides, free energies ofduplex formation were also obtained from plots of T_(m) ⁻¹ vs log₁₀(concentration). Borer et al., J. Mol. Biol., 86, 843 (1974).

EXAMPLE 26 ras Transactivation Reporter Gene System

[0359] The expression plasmid pSV2-oli, containing an activated (codon12, GGC→GTC) H-ras cDNA insert under control of the constitutive SV40promoter, was a gift from Dr. Bruno Tocque (Rhone-Poulenc Sante, Vitry,France). This plasmid was used as a template to construct, by PCR, aH-ras expression plasmid under regulation of the steroid-inducible mousemammary tumor virus (MMTV) promoter. To obtain H-ras coding sequences,the 570 bp coding region of the H-ras gene was amplified by PCR. The PCRprimers were designed with unique restriction endonuclease sites intheir 5′-regions to facilitate cloning. The PCR product containing thecoding region of the H-ras codon 12 mutant oncogene was gel purified,digested, and gel purified once again prior to cloning. Thisconstruction was completed by cloning the insert into the expressionplasmid pMAMneo (Clontech Laboratories, Calif.).

[0360] The ras-responsive reporter gene pRD053 was used to detect rasexpression. [Owen et al., Proc. Natl. Acad. Sci. U.S.A., 87, 3866(1990).

EXAMPLE 27 Northern Blot Analysis of ras Expression in vivo

[0361] The human urinary bladder cancer cell line T24 was obtained fromthe American Type Culture Collection (Rockville Md.). Cells were grownin McCoy's 5A medium with L-glutamine (GIBCO-BRL, Gaithersburg, Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of OptiMEM (GIBCO)reduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide was thenadded to the desired concentration. After 4 hours of treatment, themedium was replaced with McCoy's medium. Cells were harvested 48 hoursafter oligonucleotide treatment and RNA was isolated using a standardCsCl purification method. [Kingston in Current Protocols in MolecularBiology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A.Smith, J. G. Seidman and K. Strahl, Eds., John Wiley and Sons, NewYork.] The human epithelioid carcinoma cell line HeLa 229 was obtainedfrom the American Type Culture Collection (Bethesda, Md.). HeLa cellswere maintained as monolayers on 6-well plates in Dulbecco's ModifiedEagle's medium (DMEM) supplemented with 10 fetal bovine serum and 100U/ml penicillin. Treatment with oligonucleotide and isolation of RNAwere essentially as described above for T24 cells.

[0362] Northern hybridization: 10 μg of each RNA was electrophoresed ona 1.2% agarose/formaldehyde gel and transferred overnight to GeneBind 45nylon membrane (Pharmacia LKB, Piscataway, N.J.) using standard methods.[Kingston in Current Protocols in Molecular Biology, F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K.Strahl, Eds., John Wiley and Sons, New York.] RNA was UV-crosslinked tothe membrane. Double-stranded ³²P-labeled probes were synthesized usingthe Prime a Gene labeling kit (Promega, Madison Wis.). The ras probe wasa SalI-NheI fragment of a cDNA clone of the activated (mutant) H-rasmRNA having a GGC-to-GTC mutation at codon-12. The control probe wasG3PDH. Blots were prehybridized for 15 minutes at 68° C. with theQuickHyb hybridization solution (Stratagene, La Jolla, Calif.). Theheat-denatured radioactive probe (2.5×10⁶ counts/2 ml hybridizationsolution) mixed with 100 μl of 10 mg/ml salmon sperm DNA was added andthe membrane was hybridized for 1 hour at 68° C. The blots were washedtwice for 15 minutes at room temperature in 2×SSC/0.1% SDS and once for30 minutes at 60° C. with 0.1×SSC/0.1%SDS. Blots were autoradiographedand the intensity of signal was quantitated using an ImageQuantPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Northern blotswere first hybridized with the ras probe, then stripped by boiling for15 minutes in 0.1×SSC/0.1%SDS and rehybridized with the control G3PDHprobe to check for correct sample loading.

EXAMPLE 28 Antisense Oligonucleotide Inhibition of Proliferation ofCancer Cells

[0363] Cells were cultured and treated with oligonucleotide essentiallyas described in Example 27. Cells were seeded on 60 mm plates and weretreated with oligonucleotide in the presence of DOTMA when they reached70% confluency. Time course experiment: On day 1, cells were treatedwith a single dose of oligonucleotide at a final concentration of 100nM. The growth medium was changed once on day 3 and cells were countedevery day for 5 days, using a counting chamber. Dose-responseexperiment: Various concentrations of oligonucleotide (10, 25, 50, 100or 250 nM) were added to the cells and cells were harvested and counted3 days later. Oligonucleotides 2570, 3985 and 4690 were tested foreffects on T24 cancer cell proliferation.

EXAMPLE 29 Inhibition of PKC-α mRNA Expression by Chimeric (DeoxyGapped) 2′-O-Methyl Oligonucleotides

[0364] Oligonucleotides having SEQ ID NO: 4 were synthesized asuniformly phosphorothioate chimeric oligonucleotides having a centereddeoxy gap of varying lengths flanked by 2′-O-methylated regions. Theseoligonucleotides (500 nM concentration) were tested for effects on PKC-αmRNA levels by Northern blot analysis. Deoxy gaps of eight nucleotidesor more gave maximal reduction of PKC-α mRNA levels (both transcripts)in all cases. These oligonucleotides reduced PKC-α mRNA by approximately83% with a deoxy gap length of four nucleotides, and gave nearlycomplete reduction of PKC-α mRNA with a deoxy gap length of six or more.

[0365] The 2′-O-methyl chimeric oligonucleotides with four- orsix-nucleotide deoxy gaps have an IC₅₀ for PKC-α mRNA reduction(concentration of oligonucleotide needed to give a 50% reduction inPKC-α mRNA levels) of 200-250 nM, as did the full-deoxy oligonucleotide(all are phosphorothioates throughout). The 2′-O-methyl chimericoligonucleotide with an 8-nucleotide deoxy gap had an IC₅₀ (ofapproximately 85 nM.

[0366] Several variations of this chimeric oligonucleotide (SEQ ID NO:4) were compared for ability to lower PKC-α mRNA levels. Theseoligonucleotides are shown in Table 7. TABLE 7 Chimeric2′-O-methyl/deoxy P = S oligonucleotides bold = 2′-O-methyl; s = P = Slinkage, o = P = O linkage OLIGO SEQUENCE SEQ ID NO: 3522AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 5352AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 6996AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 4 7008AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 4 7024AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 4

[0367] Effect of these oligonucleotides on PKC-α mRNA levels is shown inFIG. 3. Oligonucleotides 7008, 3522 and 5352 show reduction of PKC-αmRNA, with 5352 being most active.

[0368] A series of 2′-O-propyl chimeric oligonucleotides was synthesizedhaving SEQ ID NO: 4. These oligonucleotides are shown in Table 8. TABLE8 Chimeric 2′-O-propyl/deoxy P = S oligonucleotides bold = 2′-O-propyl;s = P = S linkage, o = P = O linkage SEQ ID OLIGO SEQUENCE NO. 7199AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 4 7273AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 4 7294AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 4 7295AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 4

[0369] These 2′-O-propyl chimeric oligonucleotides were compared to the2′-O-methyl chimeric oligonucleotides. Oligonucleotides 7273 and 7294were more active than their 2′-O-methyl counterparts at lowering PKC-αmRNA levels. This is shown in FIGS. 4 and 5.

EXAMPLE 30 Additional Oligonucleotides Which Decrease PKC-α mRNAExpression

[0370] Additional phosphorothioate oligonucleotides targeted to thehuman PKC-α 3′ untranslated region were designed and synthesized. Thesesequences are shown in Table 9. TABLE 9 Chimeric 2′-O-propyl/deoxy P = Soligonucleotides targeted to PKC-α 3′-UTR bold = 2′-O-propyl; s = P = Slinkage, o = P = O linkage SEQ ID OLIGO SEQUENCE NO: 6632 TsTsCs TsCsGsCsTsGs GsTsGs AsGsTs TsTsC 5 6653 TsTsCsTsCsGs CsTsGs GsTsGs AsGsTsTsTsC 5 6665 ToToCoTsCsGs CsTsGs GsTsGs AsGsToToToC 5 7082 TsCsTs CsGsCsTsGsGs TsGsAs GsTsTs TsC 6 7083 TsCsTsCsGsCs TsGsGs TsGsAs GsTsTs TsC 67084 ToCoToCsGsCs TsGsGs TsGsAs GsToTo ToC 6

[0371] Oligonucleotides 6632, 6653, 7082 and 7083 are most active inreducing PKC-α mRNA levels.

EXAMPLE 31 Inhibition of c-raf Expression by Chimeric Oligonucleotides

[0372] Chimeric oligonucleotides having SEQ ID NO: 7 were designed usingthe Genbank c-raf sequence HUMRAFR (Genbank listing x03484), synthesizedand tested for inhibition of c-raf mRNA expression in T24 bladdercarcinoma cells using a Northern blot assay. These chimericoligonucleotides have central “gap” regions of 6, 8 or 10deoxynucleotides flanked by two regions of 2′-O-methyl modifiednucleotides, and are shown in Table 10. Backbones were uniformlyphosphorothioate. In a Northern blot analysis, as described in Example32, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap; ISIS6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70%inhibition of c-raf mRNA expression in T24 cells. These oligonucleotidesare preferred. The 8-deoxy gap compound (6717) showed greater than 90%inhibition and is more preferred. TABLE 10 Chimeric 2′-O-methyl P = Sdeoxy “gap” oligonucleotides bold = 2′-O-methyl OLIGO SEQUENCE Targetsite SEQ ID NO: 6720 TCCCGCCTGTGACATGCATT 3′UTR 7 6717TCCCGCCTGTGACATGCATT 3′UTR 7 6729 TCCCGCCTGTGACATGCATT 3′UTR 7

[0373] Additional chimeric oligonucleotides were synthesized having oneor more regions of 2′-O-methyl modification and uniform phosphorothioatebackbones. These are shown in Table 11. All are phosphorothioates; boldregions indicate 2′-O-methyl modified regions. TABLE 11 Chimeric2′-O-methyl P = S c-raf oligonucleotides OLIGO SEQUENCE Target site SEQID NO: 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 8 7852 TCCTCCTCCCCGCGGCGGGT 5′UTR8 7849 CTCGCCCGCTCCTCCTCCCC 5′UTR 9 7851 CTCGCCCGCTCCTCCTCCCC 5′UTR 97856 TTCTCGCCCGCTCCTCCTCC 5′UTR 10 7855 TTCTCGCCCGCTCCTCCTCC 5′UTR 107854 TTCTCCTCCTCCCCTGGCAG 3′UTR 11 7847 CTGGCTTCTCCTCCTCCCCT 3′UTR 127850 CTGGCTTCTCCTCCTCCCCT 3′UTR 12 7853 CCTGCTGGCTTCTCCTCCTC 3′UTR 13

[0374] When tested for their ability to inhibit c-raf mRNA by Northernblot analysis, ISIS 7848, 7849, 7851, 7856, 7855, 7854, 7847, and 7853gave better than 70% inhibition and are therefore preferred. Of these,7851, 7855, 7847 and 7853 gave greater than 90% inhibition and are morepreferred.

[0375] Additional chimeric oligonucleotides with various 2′modifications were prepared and tested. These are shown in Table 12. Allare phosphorothioates; bold regions indicate 2′-modified regions. TABLE12 Chimeric 2′-modified P = S c-raf oligonucleotides OLIGO SEQUENCETARGET SITE MODIFIC. SEQ ID NO: 6720 TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me7 6717 TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me 7 6729 TCCCGCCTGTGACATGCATT3′UTR 2′-O-Me 7 8097 TCTGGCGCTGCACCACTCTC 3′UTR 2′-O-Me 14 9270TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Pr 7 9058 TCCCGCCTGTGACATGCATT 3′UTR2′-F 7 9057 TCTGGCGCTGCACCACTCTC 3′UTR 2′-F 14

[0376] Of these, oligonucleotides 6720, 6717, 6729, 9720 and 9058 arepreferred. Oligonucleotides 6717, 6729, 9720 and 9058 are morepreferred.

EXAMPLE 32 Northern Blot Analysis of Inhibition of c-raf mRNA Expression

[0377] The human urinary bladder cancer cell line T24 was obtained fromthe American Type Culture Collection (Rockville, Md.). Cells were grownin McCoy's 5A medium with L-glutamine (GIBCO-BRL, Gaithersburg, Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of OptiMEMreduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide withlipofectin was then added to the desired concentration. After 4 hours oftreatment, the medium was replaced with McCoy's medium. Cells wereharvested 24 to 72 hours after oligonucleotide treatment and RNA wasisolated using a standard CsCl purification method. [Kingston in CurrentProtocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston,D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, Eds., John Wileyand Sons, New York.] Total RNA was isolated by centrifugation of celllysates over a CsCl cushion. RNA samples were electrophoresed through1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to random-primed³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as acontrol. RNA was quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

EXAMPLE 33 Oligonucleotide Inhibition of Rev Gene Expression

[0378] The chimeric oligonucleotides used in this assay are shown inTable 13 below. TABLE 13 Chimeric 2′-O-propyl/deoxy P = Soligonucleotides targeted to HIV rev gene bold = 2′-O-propyl; s = P=Slinkage; o = P=O linkage OLIGO SEQUENCE SEQ ID NO: 8907UoAoGoGoAoGoAsUsGsCsCsUsAsAoGoGoCoUoUoU 15 8908GoCoUoAoUoGoUsCsGsAsCsAsCsCoCoAoAoUoUoC 16 8909CoAoUoAoGoGoAsGsAsUsGsCsCsUoAoAoGoGoCoT 17

[0379] Transfection and Luciferase assay: 3T3 cells were maintained inDMEM with glucose, L-glutamine, sodium pyruvate and 10% fetal bovineserum (GIBCO). For all experiments, cells were seeded the previous nightat 75,000 cells/well in 6-well plates (Falcon). Transfections wereperformed using the standard CaPO₄ method. For each set of replicates,15 μg/mL of pSG5/rev plasmid, 18 μg/mL pHIVenu-luc and 2 μg/mL of Rep 6were precipitated and 200 μL of this was dripped on each well. Theprecipitate was allowed to incubate on cells for 7 hours at 37° C. Themedia was then aspirated, the cells washed once with PBS, and freshcomplete media added for overnight incubation. Following incubation, themedia was removed, cells washed with 2 mL of OPTIMEM (GIBCO) and 1 mL ofOPTIMEM containing 2.5 μg/mL of Lipofectin (GIBCO-BRL) and theoligonucleotide added. The mixture was incubated for 4 hours at 37° C.,at which point it was aspirated off the cells and complete media wasadded. Two hours after this treatment, 0.2 μM/mL of dexamethasone(Sigma) was added to all wells to allow induction of the MMTV promoterof pHIVenu-luc.

[0380] The Luciferase assay was performed 24 hours later, as follows:The wells were washed twice with PBS and the cells were harvested byscraping in 200 μL of lysis buffer (1% Triton, 25 mM glycylglycine, pH7.8, 15 mM MgSO₄, 4 mM EGTA and 1 mM DTT)> The lysate was clarified bymicrofuging for 5 minutes at 11,500 rpm in the cold. 100 μL of thelysate was then combined in a microtiter plate with 50 μL of assaybuffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 15 mMpotassium phosphate, pH 7.8, 1 mM DTT and 7.5 mM ATP). Luc detection wasperformed using a microtiter luminescent reader (Dynatech Laboratories).The reactions were started by injecting 50 μL of 1X luciferase solution(Sigma). The 1X solution was diluted in luciferin buffer (25 mMglycylglycine, pH 7.8, 15 mM MgSO₄, 4 mM EGTA and 4 mM DTT) prior to usefrom a 10X stock (10 mM luciferin in 10 mM DTT). Samples were countedfor 20 seconds. The kinetics of firefly luc light emission arecharacterized by a flash period lasting a few seconds followed by aperiod of lower light intensity emission lasting several minutes.

[0381] Rev and RRE RNA synthesis: pSGk-Rev contains the Rev geneadjacent to a T7 promoter. BglII linearized pSG5-Rev was used as a DNAtemplate for transcription with T7 RNA polymerase. A template for theproduction of RRE RNA was produced by PCR. For RNA synthesis, DNAtemplates were used at 0.2 to 1.0 mg/mL, with 5 mM each of ATP, CTP andGTP, 0.5 mM of UTP, 10 mM of DTT, 40 mM of Tris-HCl, pH 7.5, 6 mM ofMgCl₂, 4 mM of Spermidine, 500 U/mL of RNAsin at 20 U/μL, 2500 μCi/mL ofa ³²P UTP at 10 mCi/mL and 1000 U/mL of T7 RNA polymerase. The reactionwas incubated for 1 hour at 37° C. The transcription reaction wasterminated by adding formamide loading buffer and was run in adenaturing polyacrylamide gel containing 8 M urea. The RNA was elutedfrom the gel according to the procedure of Schwartz et al. (Gene, 1990,88, 197).

EXAMPLE 34 Immunoassay for Antiviral Screening

[0382] NHDF cells were seeded in 96-well culture plates at a density of15,000 cells/well in serum-free FGM. Established monolayers werepretreated with the oligonucleotide overnight in FGM prior to infection.After pretreatment, cells were rinsed thrice with fresh, prewarmed FGM,and virus in 100 μL of FGM/well was added to achieve an MOI of 0.05PFU/cell. After 2 hours of incubation at 37° C., virus was removed andfresh medium (100 μL/well) containing the oligonucleotide was added.Medium was exchanged 2 days after infection with fresh medium containingthe oligonucleotide, and 6 days after infection, the cells were fixed inabsolute ethanol and dried in preparation for antibody staining. Amodified protocol was used for some assays in which FGM was supplementedwith low levels of FBS (0.2%), and the incubation period after infectionwas shortened from 6 days to 3 days. The shorter assay eliminated theneed to exchange medium 2 days after infection. Both assays yieldedcomparable values for 50% effective concentrations (EC50s).

[0383] Fixed cells were blocked in a solution of PBS containing 2%bovine serum albumin (BSA), and mouse monoclonal antibody (1H10,supplied by Eisai Co., Ltd., Japan) was added in a 1:2000 dilution inPBS-1% BSA. The 1H10 antibody recognizes an abundant late HCMVpolypeptide approximately 65 kDa in size. Detection of bound monoclonalantibody was facilitated with biotinylated goat anti-mouseimmunoglobulin G abd streptavidin-coupled β-galactosidase (GIBCO-BRL,Gaithersburg, Md.). Chlorophenol red β-D-galactopyranoside was used as asubstrate for β-galactosidase, and activity was determined by measuringthe optical density at 575 nm of individual wells with a BioTex modelEL312e microplate reader.

[0384] The oligonucleotides used in this assay are shown in Table 14below. TABLE 14 Inhibition of CMV replication by chimeric 2′-O-methyl P=S oligonucleotides bold = 2′-O-methyl OLIGO SEQUENCE SEQ ID NO: 4325 GCGUUTGCT CTT CTT CUU GCG 18 4326 GCG UUU GCTCTT CTU CUU GCG 19

EXAMPLE 35 Diagnostic Assay for the Detection of mRNA Overexpression

[0385] Oligonucleotides are radiolabeled after synthesis by ³²P labelingat the 5′ end with polynucleotide kinase. Sambrook et al. [“MolecularCloning. A Laboratory Manual,” Cold Spring Harbor Laboratory Press,1989, Volume 2, pg. 11.31-11.32]. Radiolabeled oligonucleotide iscontacted with tissue or cell samples suspected of mRNA overexpression,such as a sample from a patient, under conditions in which specifichybridization can occur, and the sample is washed to remove unboundoligonucleotide. A similar control is maintained wherein theradiolabeled oligonucleotide is contacted with normal cell or tissuesample under conditions that allow specific hybridization, and thesample is washed to remove unbound oligonucleotide. Radioactivityreamining in the sample indicates bound oligonucleotide and isquantitated using a scintillation counter or other routine means.Comparison of the radioactivity remaining in the samples from normal anddiseased cells indicates overexpression of the mRNA of interest.

[0386] Radiolabeled oligonucleotides of the invention are also useful inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.A control with normal cell or tissue sample is also maintained. Theemulsion, when developed, yields an image of silver grains over theregions overexpressing the mRNA, which is quantitated. The extent ofmRNA overexpression is determined by comparison of the silver grainsobserved with normal and diseased cells.

[0387] Analogous assays for fluorescent detection of mRNA expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling, Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorescence microscope is used to detect the fluorescence.Comparison of the fluorescence observed in samples from normal anddiseased cells enables detection of mRNA overexpression.

EXAMPLE 36 Detection of Abnormal mRNA Expression

[0388] Tissue or cell samples suspected of expressing abnormal mRNA areincubated with a first ³²P or fluorescein-labeled oligonucleotide whichis targeted to the wild-type (normal) mRNA. An identical sample of cellsor tissues is incubated with a second labeled oligonucleotide which istargeted to the abnormal mRNA, under conditions in which specifichybridization can occur, and the sample is washed to remove unboundoligonucleotide. Label remaining in the sample indicates boundoligonucleotide and can be quantitated using a scintillation counter,fluorimeter, or other routine means. The presence of abnormal mRNA isindicated if binding is observed in the case of the second but not thefirst sample.

[0389] Double labeling can also be used with the oligonucleotides andmethods of the invention to specifically detect expression of abnormalmRNA. A single tissue sample is incubated with a first ³²P-labeledoligonucleotide which is targeted to wild-type mRNA, and a secondfluorescein-labeled oligonucleotide which is targeted to the abnormalmRNA, under conditions in which specific hybridization can occur. Thesample is washed to remove unbound oligonucleotide and the labels aredetected by scintillation counting and fluorimetry. The presence ofabnormal mRNA is indicated if the sample does not bind the ³²P-labeledoligonucleotide (i.e., is not radioactive) but does retain thefluorescent label (i.e., is fluorescent).

EXAMPLE 37 Plasma Uptake and Tissue Distribution of Oligonucleotides inMice

[0390] The following oligonucleotides were prepared:

[0391] UsGsCsAsTsCsCsCsCsCsAsGsGsCsCsAsCsCsAsT, SEQ ID NO: 20

[0392] UsGsCsAsTsCsCsCsCsAsGsGsCsCsAsCsCsAsT, SEQ ID NO: 20

[0393] UsGsCsAsTsCsCCCCAGGCsCsAsCsCsAsT, SEQ ID NO: 20

[0394] wherein bold type indicated a 2′-O-propyl substituent, “s”indicates a phosphorothioate linkage and the absence of “s” indicates aphosphodiester linkage in the respective oligonucleotides. The firstoligonucleotide is identified as Isis 3082, the second as Isis 9045 andthe third as Isis 9046 in the FIGS. 6, 7, 8 and 9. The oligonucleotideswere tritiated as per the procedure of Graham et al., Nuc. Acids Res.,1993, 16, 3737-3743.

[0395] Animals and Experimental Procedure

[0396] For each oligonucleotide studied, twenty male Balb/c mice(Charles River), weighing about 25 gm, were randomly assigned into oneof four treatment groups. Following a one-week acclimation, micereceived a single tail vein injection of ³H-radiolabeled oligonucleotide(approximately 750 nmoles/kg; ranging from 124-170 μCi/kg) administeredin phosphate buffered saline, pH 7.0. The concentration ofoligonucleotide in the dosing solution was approximately 60 μM. Oneretro-orbital bleed (at either 0.25, 0.5, 2, or 4 hours post-dose) and aterminal bleed (either 1, 3, 8 or 24 hours post-dose) was collected fromeach group. The terminal bleed was collected by cardiac puncturefollowing ketamine/xylazine anesthesia. An aliquot of each blood samplewas reserved for radioactivity determination and the remaining blood wastransferred to an EDTA-coated collection tube and centrifuged to obtainplasma. Urine and feces were collected at intervals (0-4, 4-8 and 8-24hours) from the group terminated at 24 hours.

[0397] At termination, the liver, kidneys, spleen, lungs, heart, brain,sample of skeletal muscle, portion of the small intestine, sample ofskin, pancreas, bone (both femurs containing marrow) and two lymph nodeswere collected from each mouse and weighed. Feces were weighed, thenhomogenized 1:1 with distilled water using a Brinkmann Polytronhomogenizer (Westbury, N.Y.). Plasma, tissues, urine and feceshomogenate were divided for the analysis of radioactivity by combustionand for determination of intact oligonucleotide content. All sampleswere immediately frozen on dry ice after collection and stored at −80°C. until analysis.

[0398] Analysis of Radioactivity in Plasma, Tissue, and Excreta

[0399] Plasma and urine samples were weighed directly into scintillationvials and analyzed directly by liquid scintillation counting after theaddition of 15 ml of BetaBlend (ICN Biomedicals, Costa Mesa, Calif.).All other samples (tissues, blood and homogenized feces) were weighedinto combustion boats and oxidized in a Biological Materials Oxidizer(Model OX-100; R. J. Harvey Instrument Corp., Hillsdale, N.J.). The ³H₂Owas collected in 20 ml of cocktail, composed of 15 ml of BetaBlend and 5ml of Harvey Tritium Cocktail (R. J. Harvey Instrument Corp., Hillsdale,N.J.). The combustion efficiency was determined daily by combustion ofsamples spiked with a solution of ³H-mannitol and ranged between73.9-88.3%. Liquid scintillation counting was performed using a BeckmanLS 9800 or LS 6500 Liquid Scintillation System (Beckman Instruments,Fullerton, Calif.). Samples were counted for 10 minutes with automaticquench correction. Disintergration per minute values were corrected forthe efficiency of the combustion process.

[0400] Analysis of Data

[0401] Radioactivity in samples was expressed as disintergrations perminute per gram of sample. These values were divided by the specificactivity of the radiolabel to express the data in nanomole-equivalentsof total oligonucleotide per gram of sample, then converted to percentof dose administered per organ or tissue. Assuming a tissue density of 1gm/ml, the nmole/gram data were converted to a total μM concentration.To calculate the concentration of intact oligonucleotide in plasma,liver or kidney at each time point, the mean total μM concentrationswere divided by the percent of intact oligonucleotide in the dosingsolution (82-97%), then multiplied by the mean percentage of intactoligonucleotide at each time point as determined by CGE or HPLC. Thisdata was then used for the calculation of tissue half-lives by linearregression and to compare the plasma pharmacokinetics of the differentmodified oligonucleotides. The pharmacokinetic parameters weredetermined using PCNONLIN 4.0 (Statistical Consultants, Inc., Apex,N.C.). After examination of the data, a one-compartment bolus input,first order output model (library model 1) was selected for use.

[0402] The result of the animal plasma uptake and tissue distributiontests are illustrated graphically in FIGS. 6, 7, 8 and 9. As is seen inFIG. 6, plasma concentration of each of the test oligonucleotidesdecrease from the initial injection levels to lower levels over thetwenty-four hour test period. Plasma concentrations of theoligonucleotides of the invention were maintained at levels equivalentto those of the non-conjugate bearing phosphorothioate. All of the testcompounds were taken up from the plasma to tissues as is shown in FIGS.7, 8 and 9. The compounds of the invention had different distributionbetween the various tissues. FIG. 7 shows the distribution pattern forthe control oligonucleotide, identified as ISIS 3082, a phosphorothioateoligonucleotide. FIG. 8 shows the distribution pattern for a firstcompound of the invention, an oligonucleotide, identified as ISIS 9045,having a 2′-substituent at each nucleotide. FIG. 9 shows thedistribution pattern for a further compound of the invention, a “gapmer” oligonucleotide, identified as ISIS 9046, having a 2′-substituentand phosphodiester linkages at each nucleotide at “flanking” sections ofthe oligonucleotide and 2′-deoxy, phosphorothioate nucleotides in acentral or gap region.

1 37 1 17 DNA Artificial Sequence Synthetic construct 1 ccacaccgacggcgccc 17 2 20 DNA Artificial Sequence Synthetic construct 2 cttatattccgtcatcgctc 20 3 20 DNA Artificial Sequence Synthetic construct 3tccgtcatcg ctcctcaggg 20 4 20 DNA Artificial Sequence Syntheticconstruct 4 aaaacgtcag ccatggtccc 20 5 18 DNA Artificial SequenceSynthetic construct 5 ttctcgctgg tgagtttc 18 6 17 DNA ArtificialSequence Synthetic construct 6 tctcgctggt gagtttc 17 7 20 DNA ArtificialSequence Synthetic construct 7 tcccgcctgt gacatgcatt 20 8 20 DNAArtificial Sequence Synthetic construct 8 tcctcctccc cgcggcgggt 20 9 20DNA Artificial Sequence Synthetic construct 9 ctcgcccgct cctcctcccc 2010 20 DNA Artificial Sequence Synthetic construct 10 ttctcgcccgctcctcctcc 20 11 20 DNA Artificial Sequence Synthetic construct 11ttctcctcct cccctggcag 20 12 20 DNA Artificial Sequence Syntheticconstruct 12 ctggcttctc ctcctcccct 20 13 20 DNA Artificial SequenceSynthetic construct 13 cctgctggct tctcctcctc 20 14 20 DNA ArtificialSequence Synthetic construct 14 tctggcgctg caccactctc 20 15 20 RNAArtificial Sequence Synthetic construct 15 uaggagaugc cuaaggcuuu 20 1620 RNA Artificial Sequence Synthetic construct 16 gcuaugucga cacccaauuc20 17 20 DNA Artificial Sequence Synthetic construct 17 cauaggagaugccuaaggct 20 18 21 DNA Artificial Sequence Synthetic construct 18gcguutgctc ttcttcuugc g 21 19 22 DNA Artificial Sequence Syntheticconstruct 19 gcguutgctc ttctucuuug cg 22 20 20 DNA Artificial SequenceSynthetic construct 20 ugcatccccc aggccaccat 20 21 15 DNA ArtificialSequence Synthetic construct 21 cgactatgca agtac 15 22 17 DNA ArtificialSequence Synthetic construct 22 ctcgtacctt ccggtcc 17 23 12 RNAArtificial Sequence Synthetic construct 23 gagcucccag gc 12 24 15 RNAArtificial Sequence Synthetic construct 24 cgacuaugca aguac 15 25 15 RNAArtificial Sequence Synthetic construct 25 uccagguguc cgauc 15 26 13 DNAArtificial Sequence Synthetic construct 26 tccaggccgu uuc 13 27 13 DNAArtificial Sequence Synthetic construct 27 tccaggtgtc ccc 13 28 17 RNAArtificial Sequence Synthetic construct 28 cucguaccuu ccggucc 17 29 18DNA Artificial Sequence Synthetic construct 29 ctcgtacctt tccggtcc 18 3018 RNA Artificial Sequence Synthetic construct 30 gagcauggya aggccagg 1831 18 RNA Artificial Sequence Synthetic construct 31 cucguaccuu uccggucc18 32 16 DNA Artificial Sequence Synthetic construct 32 gcgttttttttttgcg 16 33 19 DNA Artificial Sequence Synthetic construct 33cgcaaaaaaa aaaaaacgc 19 34 47 DNA Artificial Sequence Syntheticconstruct 34 acattatgct agctttttga gtaaacttgt ggggcaggag accctgt 47 3529 DNA Artificial Sequence Synthetic construct 35 gagatctgaa gcttctggatggtcagcgc 29 36 35 DNA Artificial Sequence Synthetic construct 36gagatctgaa gcttgaagac gccaaaaaca taaag 35 37 33 DNA Artificial SequenceSynthetic construct 37 acgcatctgg cgcgccgata ccgtcgacct cga 33

What is claimed is:
 1. A compound comprising a plurality ofcovalently-bound nucleosides that individually include a ribose ordeoxyribose sugar portion and a base portion, wherein: said nucleosidesare joined together by internucleoside linkages such that the baseportion of said nucleosides form a mixed base sequence; at least one ofsaid nucleosides includes a modified ribofuranosyl moiety bearing a2′-fluoro substituent; provided that at least two of said nucleosidesare 2-fluoro modified ribofuranosyl nucleosides when saidinternucleoside linkages are phosphodiester linkages.
 2. The compound ofclaim 1 having 5 to 50 nucleoside linked nucleosides.
 3. The compound ofclaim 1 wherein at least two of said nucleosides are covalently boundthrough phosphorothioate, methyl phosphoate, or phosphate alkylateinternucleoside linkages.